Oligomeric compounds comprising 4′-thionucleosides for use in gene modulation

ABSTRACT

The present invention provides modified oligomeric compounds and compositions of oligomeric compounds for use in the RNA interference pathway of gene modulation. The modified oligomeric compounds include siRNA and asRNA having at least one affinity modification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 60/503,997 filed Sep. 18, 2003, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides monomeric and oligomeric compoundscomprising 4′-thionucleosides. More particularly, the present inventionprovides oligomeric compounds and compositions comprising at least one4′-thionucleoside of the invention. In some embodiments, the oligomericcompounds and compositions of the present invention hybridize to aportion of a target RNA resulting in loss of normal function of thetarget RNA.

BACKGROUND OF THE INVENTION

Targeting disease-causing gene sequences was first suggested more thanthirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), andantisense activity was demonstrated in cell culture more than a decadelater (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75,280-284). One advantage of antisense technology in the treatment of adisease or condition that stems from a disease-causing gene is that itis a direct genetic approach that has the ability to modulate (increaseor decrease) the expression of specific disease-causing genes. Anotheradvantage is that validation of a therapeutic target using antisensecompounds results in direct and immediate discovery of the drugcandidate; the antisense compound is the potential therapeutic agent.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and modulatesgene expression activities or function, such as transcription ortranslation. The modulation of gene expression can be achieved by, forexample, target degradation or occupancy-based inhibition. An example ofmodulation of RNA target function by degradation is RNase H-baseddegradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi generally refers toantisense-mediated gene silencing involving the introduction of dsRNAleading to the sequence-specific reduction of targeted endogenous mRNAlevels. Regardless of the specific mechanism, this sequence-specificitymakes antisense compounds extremely attractive as tools for targetvalidation and gene functionalization, as well as therapeutics toselectively modulate the expression of genes involved in thepathogenesis of malignancies and other diseases.

Antisense compounds have been employed as therapeutic agents in thetreatment of disease states in animals, including humans. Antisenseoligonucleotide drugs are being safely and effectively administered tohumans in numerous clinical trials. In 1998, the antisense compound,Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc.,Carlsbad, Calif.) was the first antisense drug to achieve marketingclearance from the U.S. Food and Drug Administration (FDA), and iscurrently used in the treatment of cytomegalovirus (CMV)-inducedretinitis in AIDS patients. A New Drug Application (NDA) for Genasense™(oblimersen sodium; developed by Genta, Inc., Berkeley Heights, N.J.),an antisense compound which targets the Bcl-2 mRNA overexpressed in manycancers, was accepted by the FDA. Many other antisense compounds are inclinical trials, including those targeting c-myc (NeuGene® AVI-4126, AVIBioPharma, Ridgefield Park, N.J.), TNF-alpha (ISIS 104838, developed byIsis Pharmaceuticals, Inc.), VLA4 (ATL1102, Antisense Therapeutics Ltd.,Toorak, Victoria, Australia) and DNA methyltransferase (MG98, developedby MGI Pharma, Bloomington, Minn.), to name a few.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy.

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications.

Consequently, there remains a long-felt need for agents thatspecifically regulate gene expression via antisense mechanisms.Disclosed herein are antisense compounds useful for modulating geneexpression pathways, including those relying on mechanisms of actionsuch as RNaseH, RNAi and dsRNA enzymes, as well as other antisensemechanisms based on target degradation or target occupancy. One havingskill in the art, once armed with this disclosure will be able, withoutundue experimentation, to identify, prepare and exploit antisensecompounds for these uses.

In many species, introduction of double-stranded RNA (dsRNA) inducespotent and specific gene silencing. This phenomenon occurs in bothplants and animals and has roles in viral defense and transposonsilencing mechanisms. This phenomenon was originally described more thana decade ago by researchers working with the petunia flower. Whiletrying to deepen the purple color of these flowers, Jorgensen et al.introduced a pigment-producing gene under the control of a powerfulpromoter. Instead of the expected deep purple color, many of the flowersappeared variegated or even white. Jorgensen named the observedphenomenon “cosuppression”, since the expression of both the introducedgene and the homologous endogenous gene was suppressed (Napoli et al.,Plant Cell, 1990, 2, 279-289; Jorgensen et al., Plant Mol. Biol., 1996,31, 957-973).

Cosuppression has since been found to occur in many species of plants,fungi, and has been particularly well characterized in Neurosporacrassa, where it is known as “quelling” (Cogoni et al., Genes Dev. 2000,10, 638-643; and Guru, Nature, 2000, 404, 804-808).

The first evidence that dsRNA could lead to gene silencing in animalscame from work in the nematode, Caenorhabditis elegans. In 1995,researchers Guo and Kemphues were attempting to use antisense RNA toshut down expression of the par-1 gene in order to assess its function.As expected, injection of the antisense RNA disrupted expression ofpar-1, but quizzically, injection of the sense-strand control alsodisrupted expression (Guo et al., Cell, 1995, 81, 611-620). This resultwas a puzzle until Fire et al. injected dsRNA (a mixture of both senseand antisense strands) into C. elegans. This injection resulted in muchmore efficient silencing than injection of either the sense or theantisense strands alone. Injection of just a few molecules of dsRNA percell was sufficient to completely silence the homologous gene'sexpression. Furthermore, injection of dsRNA into the gut of the wormcaused gene silencing not only throughout the worm, but also in firstgeneration offspring (Fire et al., Nature, 1998, 391, 806-811).

The potency of this phenomenon led Timmons and Fire to explore thelimits of the dsRNA effects by feeding nematodes bacteria that had beenengineered to express dsRNA homologous to the C. elegans unc-22 gene.Surprisingly, these worms developed an unc-22 null-like phenotype(Timmons and Fire, Nature 1998, 395, 854; and Timmons et al., Gene,2001, 263, 103-112). Further work showed that soaking worms in dsRNA wasalso able to induce silencing (Tabara et al., Science, 1998, 282,430-431). PCT publication WO 01/48183 discloses methods of inhibitingexpression of a target gene in a nematode worm involving feeding to theworm a food organism which is capable of producing a double-stranded RNAstructure having a nucleotide sequence substantially identical to aportion of the target gene following ingestion of the food organism bythe nematode, or by introducing a DNA capable of producing thedouble-stranded RNA structure (Bogaert et al., 2001).

The posttranscriptional gene silencing defined in C. elegans resultingfrom exposure to double-stranded RNA (dsRNA) has since been designatedas RNA interference (RNAi). This term has come to generalize all formsof gene silencing involving dsRNA leading to the sequence-specificreduction of endogenous targeted mRNA levels; unlike co-suppression, inwhich transgenic DNA leads to silencing of both the transgene and theendogenous gene. Introduction of exogenous double-stranded RNA (dsRNA)into C. elegans has been shown to specifically and potently disrupt theactivity of genes containing homologous sequences. Montgomery et al.suggests that the primary interference effects of dsRNA arepost-transcriptional; this conclusion being derived from examination ofthe primary DNA sequence after dsRNA-mediated interference a finding ofno evidence of alterations followed by studies involving alteration ofan upstream operon having no effect on the activity of its downstreamgene. These results argue against an effect on initiation or elongationof transcription. Finally, they observed by in situ hybridization, thatdsRNA-mediated interference produced a substantial, although notcomplete, reduction in accumulation of nascent transcripts in thenucleus, while cytoplasmic accumulation of transcripts was virtuallyeliminated. These results indicate that the endogenous mRNA is theprimary target for interference and suggest a mechanism that degradesthe targeted mRNA before translation can occur. It was also found thatthis mechanism is not dependent on the SMG system, an mRNA surveillancesystem in C. elegans responsible for targeting and destroying aberrantmessages. The authors further suggest a model of how dsRNA mightfunction as a catalytic mechanism to target homologous mRNAs fordegradation. (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507).

Recently, the development of a cell-free system from syncytialblastoderm Drosophila embryos that recapitulates many of the features ofRNAi has been reported. The interference observed in this reaction issequence specific, is promoted by dsRNA but not single-stranded RNA,functions by specific mRNA degradation, and requires a minimum length ofdsRNA. Furthermore, preincubation of dsRNA potentiates its activitydemonstrating that RNAi can be mediated by sequence-specific processesin soluble reactions (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

In subsequent experiments, Tuschl et al, using the Drosophila in vitrosystem, demonstrated that 21- and 22-nt RNA fragments are thesequence-specific mediators of RNAi. These fragments, which they termedshort interfering RNAs (siRNAs) were shown to be generated by an RNaseIII-like processing reaction from long dsRNA. They also showed thatchemically synthesized siRNA duplexes with overhanging 3′ ends mediateefficient target RNA cleavage in the Drosophila lysate, and that thecleavage site is located near the center of the region spanned by theguiding siRNA. In addition, they suggest that the direction of dsRNAprocessing determines whether sense or antisense target RNA can becleaved by the siRNA-protein complex (Elbashir et al., Genes Dev., 2001,15, 188-200). Further characterization of the suppression of expressionof endogenous and heterologous genes caused by the 21-23 nucleotidesiRNAs have been investigated in several mammalian cell lines, includinghuman embryonic kidney (293) and HeLa cells (Elbashir et al., Nature,2001, 411, 494-498).

Most recently, Tijsterman et al. have shown that, in fact,single-stranded RNA oligomers of antisense polarity can be potentinducers of gene silencing. As is the case for co-suppression, theyshowed that antisense RNAs act independently of the RNAi genes rde-1 andrde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD boxRNA helicase, mut-14. According to the authors, their data favor thehypothesis that gene silencing is accomplished by RNA primer extensionusing the mRNA as template, leading to dsRNA that is subsequentlydegraded suggesting that single-stranded RNA oligomers are ultimatelyresponsible for the RNAi phenomenon (Tijsterman et al., Science, 2002,295, 694-697).

Several recent publications have described the structural requirementsfor the dsRNA trigger required for RNAi activity. Recent reports haveindicated that ideal dsRNA sequences are 21 nt in length containing 2 nt3′-end overhangs (Elbashir et al, EMBO, 2001, 20, 6877-6887; and SabineBrantl, Biochimica et Biophysica Acta, 2002, 1575, 15-25). In thissystem, substitution of the 4 nucleosides from the 3′-end with2′-deoxynucleosides has been demonstrated to not affect activity. On theother hand, substitution with 2′deoxynucleosides or 2′-OMe-nucleosidesthroughout the sequence (sense or antisense) was shown to be deleteriousto RNAi activity.

Investigation of the structural requirements for RNA silencing in C.elegans has demonstrated modification of the internucleotide linkage(phosphorothioate) to not interfere with activity (Parrish et al.,Molecular Cell, 2000, 6, 1077-1087). It was also shown by Parrish etal., that chemical modification like 2′-amino or 5′-iodouridine are welltolerated in the sense strand but not the antisense strand of the dsRNAsuggesting differing roles for the 2 strands in RNAi. Base modificationsuch as guanine to inosine (where one hydrogen bond is lost) has beendemonstrated to decrease RNAi activity independently of the position ofthe modification (sense or antisense). Same “position independent” lossof activity has been observed following the introduction of mismatchesin the dsRNA trigger. Some types of modifications, for exampleintroduction of sterically demanding bases such as 5-iodoU, have beenshown to be deleterious to RNAi activity when positioned in theantisense strand, whereas modifications positioned in the sense strandwere shown to be less detrimental to RNAi activity. As was the case forthe 21 nt dsRNA sequences, RNA-DNA heteroduplexes did not serve astriggers for RNAi. However, dsRNA containing 2′-F-2′-deoxynucleosidesappeared to be efficient in triggering RNAi response independent of theposition (sense or antisense) of the 2′-F-2′-deoxynucleosides.

In one experiment the reduction of gene expression was studied usingelectroporated dsRNA and a 25 mer morpholino in post implantation mouseembryos (Mellitzer et al., Mehanisms of Development, 2002, 118, 57-63).The morpholino oligomer did show activity but was not as effective asthe dsRNA.

A number of PCT applications have recently been published that relate tothe RNAi phenomenon. These include: PCT publication WO 00/44895; PCTpublication WO 00/49035; PCT publication WO 00/63364; PCT publication WO01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCTpublication WO 00/44914; PCT publication WO 01/29058; and PCTpublication WO 01/75164.

U.S. Pat. Nos. 5,898,031 and 6,107,094, each of which is commonly ownedwith this application and each of which is herein incorporated byreference, describe certain oligonucleotide having RNA like properties.When hybridized with RNA, these olibonucleotides serve as substrates fora dsRNase enzyme with resultant cleavage of the RNA by the enzyme.

In another recently published paper (Martinez et al., Cell, 2002, 110,563-574) it was shown that double stranded as well as single strandedsiRNA resides in the RNA-induced silencing complex (RISC) together withelF2C1 and elf2C2 (human GERp950 Argonaute proteins. The activity of5′-phosphorylated single stranded siRNA was comparable to the doublestranded siRNA in the system studied. In a related study, the inclusionof a 5′-phosphate moiety was shown to enhance activity of siRNAs in vivoin Drosophilia embryos (Boutla et al., Curr. Biol., 2001, 11,1776-1780). In another study, it was reported that the 5′-phosphate wasrequired for siRNA function in human HeLa cells (Schwarz et al.,Molecular Cell, 2002, 10, 537-548).

In yet another recently published paper (Chiu et al., Molecular Cell,2002, 10, 549-561) it was shown that the 5′-hydroxyl group of the siRNAis essential as it is phosphorylated for activity while the 3′-hydroxylgroup is not essential and tolerates substitute groups such as biotin.It was further shown that bulge structures in one or both of the senseor antisense strands either abolished or severely lowered the activityrelative to the unmodified siRNA duplex. Also shown was severe loweringof activity when psoralen was used to cross link an siRNA duplex.

Phosphorus protecting groups such as SATE ((S-acetyl-2-thioethyl)phosphate) have been used to block the phosphorus moiety of individualnucleotides and the internucleotide phosphorus linking moietys ofoligonucleotides. These groups have also been used in biological systemsto afford deprotected oligonucleotides intracellularly due to the actionof intercellular esterases. Such groups are disclosed in PCTpublications WO 96/07392, WO 93/24510, WO 94/26764 and U.S. Pat. No.5,770,713.

One group of researchers has been studying the synthesis and certainproperties of 4′-thio-containing compounds and have published theirresults (Nucleosides & Nucleotides, 1999, 18(6 & 7), 1423-1424;Antisense Research and Development, 1995, 5(3), 167-74; ACS SymposiumSeries, 1994, 580 (Carbohydrate Modifications in Antisense Research),68-79; and Nucleosides & Nucleotides, 1995, 14(3-5), 1027-30).

Another paper describes the properties of oligodeoxynucleotidescontaining deoxy 4′-thionucleotides (Nucleic Acids Research, 1996,24(21), 4117-4122).

The stereosynthesis of 4′-thioribonucleosides utilizing Pummererreaction has been described by another group of researchers (NucleicAcids Symposium Series, 1998, 39, 21-22; and J. American ChemicalSociety, 2000, 122(30), 7233-7243).

Like the RNAse H pathway, the RNA interference pathway of antisensemodulation of gene expression is an effective means for modulating thelevels of specific gene products and may therefore prove to be uniquelyuseful in a number of therapeutic, diagnostic, and research applicationsinvolving gene silencing. The present invention therefore furtherprovides oligomeric compounds useful for modulating gene expressionpathways, including those relying on an antisense mechanism of actionsuch as RNA interference and dsRNA enzymes as well as non-antisensemechanisms. One having skill in the art, once armed with this disclosurewill be able, without undue experimentation, to identify additionaloligomeric compounds for these and other uses.

SUMMARY OF THE INVENTION

The present invention provides compounds having of formula (I):

wherein:

-   -   T₁ is H, a protecting group, an activated phosphorus group, or,        or -L_(s)-SS, wherein L_(s) is a linking moiety and SS is a        solid support medium;    -   T₂ is H, a protecting group, an activated phosphorus group, or        -L_(s)-SS, wherein L_(s) is a linking moiety and SS is a solid        support medium;    -   Bx is hydrogen or a nucleobase;    -   X is halogen, amino, azido, substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, substituted        or unsubstituted C₂-C₁₂ alkynyl, substituted or unsubstituted        alkoxy, substituted or unsubstituted —O—C₂-C₁₂ alkenyl, or        substituted or unsubstituted —O—C₂-C₁₂ alkynyl, or X is a group        of formula Ia or Ib:

-   -   wherein:    -   R_(b) is O, S or NH;    -   R_(d) is a single bond, O, S or C(═O);    -   R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),        N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula Ic;

-   -   R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;    -   R_(r) is —R_(x)—R_(y);    -   each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,        C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl,        substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or        unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a        chemical functional group or a conjugate group, wherein the        substituent groups are selected from hydroxyl, amino, alkoxy,        carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,        alkyl, aryl, alkenyl and alkynyl;    -   or optionally, R_(u) and R_(v), together form a phthalimido        moiety with the nitrogen atom to which they are attached;    -   each R_(w) is, independently, substituted or unsubstituted        C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   R_(k) is hydrogen, an amino protecting group or —R_(x)—R_(y);    -   R_(p) is hydrogen, an amino protecting group or —R_(x)—R_(y);    -   R_(x) is a bond or a linking moiety;    -   R_(y) is a chemical functional group, a conjugate group or a        solid support medium;    -   each R_(m) and R_(n) is, independently, H, an amino protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where said acyl is an acid        amide or an ester;    -   or R_(m) and R_(n), together, are an amino protecting group, are        joined in a ring structure that optionally includes an        additional heteroatom selected from N and O or are a chemical        functional group;    -   R_(i) is OR_(z), SR_(z), or N(R_(z))₂;    -   each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);    -   R_(f), R_(g) and R_(h) comprise a ring system having from about        4 to about 7 carbon atoms or having from about 3 to about 6        carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are        selected from oxygen, nitrogen and sulfur and wherein said ring        system is aliphatic, unsaturated aliphatic, aromatic, or        saturated or unsaturated heterocyclic;    -   R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,        alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to        about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,        N(R_(k))(R_(m))OR_(k), halo, SR_(k) or CN;    -   m_(a) is 1 to about 10;    -   each mb is, independently, 0 or 1;    -   mc is 0 or an integer from 1 to 10;    -   md is an integer from 1 to 10;    -   me is from 0, 1 or 2; and    -   provided that when mc is 0, md is greater than 1.

In another embodiment of the present invention, oligomeric compounds aredisclosed having at least one moiety of formula (II):

wherein Bx and X are defined herein and L₁ and L₂ are an internucleosidelinkage as defined herein. Preferred internucleoside linkages for theoligomers of the present invention include, but are not limited to,phosphodiester linkages, phosphothioate linkages, and mixtures thereof.Preferred oligomers of the present invention include, but are notlimited to, oligomers 8-80 nucleotides, 8-50 nucleotides, 8-30nucleotides, 10-30 nucleotides, 15-30 nucleotides, 15-25 nucleotides,and the like.

Preferred X 2′-substituents include, but are not limited to: OH, F,O-alkyl (e.g. O-methyl), S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl or alkynyl, respectively. Particularlypreferred are O[(CH₂)_(g)O]_(h)CH₃, O(CH₂)_(g)OCH₃, O(CH₂)_(g)NH₂,O(CH₂)_(g)CH₃, O(CH₂)_(g)ONH₂, and O(CH₂)_(g)ON[(CH₂)_(g)H]₂, where gand h are from 1 to about 10. Other preferred oligonucleotides compriseone of the following at the 2′ position: C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkarylor O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heteroaromatic, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligonucleotide, or a group for improving thepharmacodynamic properties of an oligonucleotide, and other substituentshaving similar properties. In other embodiments, X can include2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504). Infurther embodiments of the invention, X can include2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples delineated herein.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl(2′-CH₂—CH═CH₂),2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modificationmay be in the arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.

Particularly useful sugar substituent groups includeO[(CH₂)_(g)O]_(h)CH₃, O(CH₂)_(g)OCH₃, O(CH₂)_(g)NH₂, O(CH₂)_(g)CH₃,O(CH₂)_(g)ONH₂, and O(CH₂)_(g)ON[(CH₂)_(g)H)]₂, where g and h are from 1to about 10.

In one embodiment of the present invention, R₂ is H or hydroxyl and R₁is allyl, amino, azido, O—CH₃, O—CH₂CH₂—O—CH₃,O—(CH₂)_(ma)—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n));

-   -   wherein:        -   each R_(m) and R_(n) is, independently, H, an amino            protecting group, substituted or unsubstituted C₁-C₁₀ alkyl,            substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or            unsubstituted C₂-C₁₀ alkynyl, wherein the substituent groups            are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,            phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,            alkenyl, alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl            where said acyl is an acid amide or an ester; and    -   ma is from 1 to about 10.

A further embodiment of the present invention includes phorphoramiditesof the nucleosides described by formula I, wherein one of T₁ or T₂ is anactivated phosphorous group.

In yet another embodiment of the present invention are solid supportbound nucleosides, wherein for formula I, one of T₁ or T₂ is -L_(s)-SS,wherein L_(s) is a linking moiety and SS is a support medium.

In another embodiment of the present invention, compositions aredisclosed comprising a first oligonucleotide and a secondoligonucleotide, wherein:

-   -   at least a portion of said first oligonucleotide is capable of        hybridizing with at least a portion of said second        oligonucleotide,    -   at least a portion of said first oligonucleotide is        complementary to and capable of hybridizing to a selected target        nucleic acid, and    -   at least one of said first or said second oligonucleotides        includes at least one nucleoside having a modification        comprising a 4′-thionucleoside of formula II:

wherein Bx, L₁ and L₂ are defined herein.

In one embodiment, the composition comprises first and secondoligonucleotides that are a complementary pair of siRNAoligonucleotides. In another embodiment the composition comprises firstand second oligonucleotides that are an antisense/sense pair ofoligonucleotides.

In one embodiment of the present invention, each of the first and secondoligonucleotides comprising the composition has from 10 to about 40nucleosides in length. In another embodiment of the present invention,each of the first and second oligonucleotides comprising the compositionhas from 18 to about 30 nucleosides in length. In a further embodimentof the present invention, each of the first and second oligonucleotidescomprising the composition has from 18 to about 24 nucleosides inlength.

In one embodiment of the present invention, the first oligonucleotide isan antisense oligonucleotide. In another embodiment of the presentinvention, the second oligonucleotide is a sense oligonucleotide. In afurther embodiment of the present invention, the second oligonucleotidehas a plurality of ribose nucleotide units.

In one embodiment of the present invention, the first oligonucleotideincludes the nucleoside having the modification.

In one embodiment of the present invention, a process for thepreparation of1,4-anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)₄-sulfinyl-D-ribitolis disclosed comprising:

-   -   preparing an oxidizing mixture by adding diethyl-L-tartrate to a        solution of Ti(IV) isopropoxide in a suitable solvent followed        by the addition of a suitable peroxide in said suitable solvent;    -   treating        1,4-anhydro-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-D-ribitol        in a suitable solvent with said oxidizing mixture to form        1,4-anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-sulfinyl-D-ribitol.

Yet another embodiment of the present invention includes processes ofmaking any of the compounds of the present invention delineated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. depicts representative PTEN siRNA constructs of the presentinvention; and

FIG. 2 depicts the reduction of human PTEN mRNA in HeLa cells achievedwith select siRNAs of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel 4′-thionucleosides, single anddouble stranded oligomeric compounds prepared therefrom. The4′-thionucleosides of the present invention are useful for chemicallyand enzymatically stabilizing oligomeric compounds in which they areincorporated, especially oligoribonucleotides. It is further believedthat the 4′-thionucleosides of the present invention have the desiredconformation to enhance the substrate specificity for mechanisms ofaction that utilize siRNA duplexes, micro RNAs and asRNA single strandedcompounds. The oligomers of the present invention may also be useful asprimers and probes in diagnostic applications.

The 4′-thionucleosides of the present invention are useful in a varietyof motifs including but not limited to hemimers, gapmers, uniform,alternating with other chemistries. In conjunction with these motifs awide variety of linkages can also be used including but not limited tophosphodiester and phosphorothioate linkages used uniformly or incombinations. The positioning of 4′-thio nucleosides and the use oflinkage strategies can be easily optimized for the best activity for aparticular target.

The 4′-thionucleosides of the present invention retain, for the mostpart, the chemical reactivity and steric positioning of functionalgroups found in natural ribonucleosides having similar conformations tocorresponding ribonucleosides. Hence, when incorporated intooligonucleotides, the resulting 4′-thioRNA has a similar conformation tonatural RNA with an expected increase in stability.

NMR experiments indicate a predominantly anti-conformation of thenucleobase as in unmodified ribonucleosides. Importantly the pKa valuesof nucleobases in 4′-thionucleosides are unchanged. This results inretention of the expected hybridization properties found in native RNA.Therefore, the 4′-thionucleosides are expected to have comparablehybridization and Tm properties with enhanced nuclease stability ascompared to native phosphodiester linked RNA. The phosphodiester4′-thionucleoside modified oligomeric compounds of the present inventionare expected to have enhanced protein-binding, alteredtissue-distribution, and pK properties.

4′-thionucleosides of the present invention are compounds of formula I,wherein X is selected from halogallyl, amino, azido, O-allyl, O—CH₃,O—CH₂CH₂—O—CH₃, O—(CH₂—CH₂—O—N(R_(m))(R_(n)) orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

Oligomeric compounds and compositions having two strands have at leastone modified nucleoside wherein X is selected from is halogen especiallyfluoro, alkyl especially methyl, alkenyl especially allyl, amino,substituted amino, azido, alkoxy especially —O-methyl or alkoxysubstituted alkoxy especially —O—CH₂CH₂—O—CH₃.

Other suitable X substituent groups are aminooxy, substituted aminooxy,—O-acetamido, substituted —O-acetamido (—O—CH₂C(═O)NR_(m)R_(n)),aminoethyloxyethoxy, substitutedaminoethyloxyethoxy(—O—CH₂CH₂—O—CH₂CH₂—NR_(m)R_(n)), aminooxyethyloxyand substituted aminooxyethyloxy(—O—CH₂CH₂—O—NR_(m)R_(n)).

Other suitable X substituents are selected from fluoro, allyl, amino,azido, O—CH₃, O—CH₂CH₂—O—CH₃, O—(CH₂)_(ma)—O—N(R_(m))(R_(n)) orO—CH₂—C(═O)—N(R_(m))(R_(n)), wherein:

-   -   each R_(m) and R_(n) is, independently, H, an amino protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where said acyl is an acid        amide or an ester; and    -   ma is from 1 to about 10.

The present invention also discloses a novel process for the preparationof1,4-anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-sulfinyl-D-ribitolcomprising:

-   -   preparing an oxidizing mixture by adding diethyl-L-tartrate to a        solution of Ti(IV) isopropoxide in a suitable solvent followed        by the addition of a suitable peroxide in a suitable solvent;    -   treating        1,4-anhydro-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-D-ribitol        in a suitable solvent with said oxidizing mixture to form        1,4-anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyl        disiloxane-1,3-diyl)-4-sulfinyl-D-ribitol.

The preparation of ribonucleotides and oligomeric compounds having atleast one ribonucleoside incorporated and all the possibleconfigurations falling in between these two extremes are encompassed bythe present invention. The corresponding oligomeric compounds can behybridized to further oligomeric compounds includingoligoribonucleotides having regions of complementarity to formdouble-stranded (duplexed) oligomeric compounds. Such double strandedoligonucleotide moieties have been shown in the art to modulate targetexpression and regulate translation as well as RNA processsing via anantisense mechanism. Moreover, the double-stranded moieties may besubject to chemical modifications (Fire et al., Nature, 1998, 391,806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene,2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431;Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507;Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). Forexample, such double-stranded moieties have been shown to inhibit thetarget by the classical hybridization of antisense strand of the duplexto the target, thereby triggering enzymatic degradation of the target(Tijsterman et al., Science, 2002, 295, 694-697).

The methods of preparing oligomeric compounds of the present inventioncan also be applied in the areas of drug discovery and targetvalidation. The present invention comprehends the use of the oligomericcompounds and targets identified herein in drug discovery efforts toelucidate relationships that exist between proteins and a disease state,phenotype, or condition. These methods include detecting or modulating atarget peptide comprising contacting a sample, tissue, cell, or organismwith the oligomeric compounds of the present invention, measuring thenucleic acid or protein level of the target and/or a related phenotypicor chemical endpoint at some time after treatment, and optionallycomparing the measured value to a non-treated sample or sample treatedwith a further oligomeric compound of the invention. These methods canalso be performed in parallel or in combination with other experimentsto determine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature (2001), 411,494-498; Nishikura et al., Cell (2001), 107, 415-416; and Bass et al.,Cell (2000), 101, 235-238.)

DEFINITIONS

General Chemistry

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.All terms appearing herein which are not specifically defined, shall beaccorded the meaning that one of ordinary skill in the relevant artwould attached to said term.

The terms “C₁-C₁₂ alkyl,” as used herein, refer to saturated, straight-or branched-chain hydrocarbon radicals containing between one and three,one and twelve, or one and six carbon atoms, respectively. Examples ofC₁-C₁₂ alkyl radicals include, but are not limited to, ethyl, propyl,isopropyl, n-hexyl, octyl, decyl, dodecyl radicals.

An “aliphatic group” is an acyclic, non-aromatic moiety that may containany combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen,nitrogen, sulfur or other atoms, and optionally contain one or moreunits of unsaturation, e.g., double and/or triple bonds. An aliphaticgroup may be straight chained, or branched and preferably containsbetween about 1 and about 24 carbon atoms, more typically between about1 and about 12 carbon atoms. In addition to aliphatic hydrocarbongroups, aliphatic groups include, for example, polyalkoxyalkyls, such aspolyalkylene glycols, polyamines, and polyimines, for example. Suchaliphatic groups may be further substituted.

Suitable substituents of the present invention include, but are notlimited to, —F, —Cl, —Br, —I, —OH, protected hydroxy, aliphatic ethers,aromatic ethers, oxo, azido, —NO₂, —CN, —C₁-C₁₂-alkyl optionallysubstituted with halogen (such as perhaloalkyls), C₂-C₁₂-alkenyloptionally substituted with halogen, —C₂-C₁₂-alkynyl optionallysubstituted with halogen, —NH₂, protected amino, —NH—C₁-C₁₂-alkyl,—NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkenyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl,—NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino, -diarylamino,-diheteroarylamino, —O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl,—O—C₂-C₁₂-alkynyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl,—O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl,—C(O)—C₂-C₁₂-alkynyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkynyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl, —CO₂—C₁-C₁₂-alkyl,—CO₂—C₂-C₁₂-alkenyl, —CO₂—C₂-C₁₂-alkynyl, —CO₂—C₃-C₁₂-cycloalkyl,—CO₂-aryl, —CO₂-heteroaryl, —CO₂-heterocycloalkyl, —OCO₂—C₁-C₁₂-alkyl,—OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkynyl, —OCO₂—C₃-C₁₂-cycloalkyl,—OCO₂-aryl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂,—OCONH—C₁-C₁₂-alkyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkynyl,—OCONH—C₃-C₁₂-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl,—OCONH-heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl,—NHC(O)—C₂-C₁₂-alkynyl, —NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl,—NHC(O)-heteroaryl, —NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl,—NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₂-C₁₂-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl,—NHCO₂-aryl, —NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkynyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkynyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkynyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl —SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,-heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl. It is understood that the aryls, heteroaryls, alkylsand the like can be further substituted.

The terms “C₂-C₁₂ alkenyl” or “C₂-C₆ alkenyl,” as used herein, denote amonovalent group derived from a hydrocarbon moiety containing from twoto twelve or two to six carbon atoms having at least one carbon-carbondouble bond by the removal of a single hydrogen atom. Alkenyl groupsinclude, but are not limited to, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, alkadienes and the like.

The term “substituted alkenyl,” as used herein, refers to a “C₂-C₁₂alkenyl” or “C₂-C₆ alkenyl” group as previously defined, substituted byone, two, three or more substituents.

The terms “C₂-C₁₂ alkynyl” or “C₂-C₆ alkynyl,” as used herein, denote amonovalent group derived from a hydrocarbon moiety containing from twoto twelve or two to six carbon atoms having at least one carbon-carbontriple bond by the removal of a single hydrogen atom. Representativealkynyl groups include, but are not limited to, for example, ethynyl,1-propynyl, 1-butynyl, and the like.

The term “substituted alkynyl,” as used herein, refers to a “C₂-C₁₂alkynyl” or “C₂-C₆ alkynyl” group as previously defined, substituted byone, two, three or more substituents.

The term “alkoxy,” as used herein, refers to an aliphatic group, aspreviously defined, attached to the parent molecular moiety through anoxygen atom. Examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,n-pentoxy, neopentoxy and n-hexoxy.

The term “substituted alkoxy,” as used herein, is an alkoxy group asdefined herein substituted with one, two, three or more substituents aspreviously defined.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “aryl” or “aromatic” as used herein, refer to a mono- orbicyclic carbocyclic ring system having one or two aromatic ringsincluding, but not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, idenyl and the like.

The terms “substituted aryl” or “substituted aromatic,” as used herein,refer to an aryl or aromatic group substituted by one, two, three ormore substituents.

The term “arylalkyl,” as used herein, refers to an aryl group attachedto the parent compound via a C₁-C₃ alkyl or C₁-C₆ alkyl residue.Examples include, but are not limited to, benzyl, phenethyl and thelike.

The term “substituted arylalkyl,” as used herein, refers to an arylalkylgroup, as previously defined, substituted by one, two, three or moresubstituents.

The terms “heteroaryl” or “heteroaromatic,” as used herein, refer to amono-, bi-, or tri-cyclic aromatic radical or ring having from five toten ring atoms of which at least one ring atom is selected from S, O andN; zero, one or two ring atoms are additional heteroatoms independentlyselected from S, O and N; and the remaining ring atoms are carbon,wherein any N or S contained within the ring may be optionally oxidized.Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. The heteroaromatic ring may be bonded to the chemical structurethrough a carbon or hetero atom.

The terms “substituted heteroaryl” or “substituted heteroaromatic,” asused herein, refer to a heteroaryl or heteroaromatic group, substitutedby one, two, three, or more substituents.

The term “alicyclic,” as used herein, denotes a monovalent group derivedfrom a monocyclic or bicyclic saturated carbocyclic ring compound by theremoval of a single hydrogen atom. Examples include, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1]heptyl, and bicyclo [2.2.2] octyl.

The term “substituted alicyclic,” as used herein, refers to an alicyclicgroup substituted by one, two, three or more substituents.

The term “heterocyclic,” as used herein, refers to a non-aromatic ring,comprising three or more ring atoms, or a bi- or tri-cyclic group fusedsystem, where (i) each ring contains between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, (ii) each5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms mayoptionally be oxidized, (iv) the nitrogen heteroatom may optionally bequaternized, (iv) any of the above rings may be fused to a benzene ring,and (v) the remaining ring atoms are carbon atoms which may beoptionally oxo-substituted. Representative heterocycloalkyl groupsinclude, but are not limited to, [1,3]dioxolane, pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, and tetrahydrofuryl.

The term “substituted heterocyclic,” as used herein, refers to aheterocyclic group, as previously defined, substituted by one, two,three or more substituents.

The term “heteroarylalkyl,” as used herein, to an heteroaryl groupattached to the parent compound via a C₁-C₃ alkyl or C₁-C₆ alkylresidue. Examples include, but are not limited to, pyridinylmethyl,pyrimidinylethyl and the like.

The term “substituted heteroarylalkyl,” as used herein, refers to aheteroarylalkyl group, as previously defined, substituted by independentreplacement of one, two, or three or more substituents.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl).

The term “dialkylamino” refers to a group having the structure —N(C₁-C₁₂alkyl)(C₁-C₁₂ alkyl) and cyclic amines. Examples of dialkylamino are,but not limited to, dimethylamino, diethylamino, methylethylamino,piperidino, morpholino and the like.

The term “alkoxycarbonyl” represents an ester group, i.e., an alkoxygroup, attached to the parent molecular moiety through a carbonyl groupsuch as methoxycarbonyl, ethoxycarbonyl, and the like.

The term “carboxaldehyde,” as used herein, refers to a group of formula—CHO.

The term “carboxy,” as used herein, refers to a group of formula —COOH.

The term “carboxamide,” as used herein, refers to a group of formula—C(O)NH(C₁-C₁₂ alkyl) or —C(O)N(C₁-C₁₂ alkyl) (C₁-C₁₂ alkyl), —C(O)NH₂,NHC(O)(C₁-C₁₂ alkyl), N(C₁-C₁₂ alkyl)C(O)(C₁-C₁₂ alkyl) and the like.

The term “protecting groups,” as used herein, refers to a labilechemical moiety which is known in the art to protect a hydroxyl or aminogroup against undesired reactions during synthetic procedures. Aftersaid synthetic procedure(s) the blocking group as described herein maybe selectively removed. Blocking groups as known in the are describedgenerally in T. H. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of hydroxyl blocking groups include, but are limited to,benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl,isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,triphenylmethyl(trityl), 4,4′-dimethoxytriphenylmethyl (DMT),substituted or unsubstituted pixyl, tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxyl blocking groups for the present invention are DMT andsubstituted or unsubstituted pixyl.

Amino blocking groups as known in the are described generally in T. H.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rdedition, John Wiley & Sons, New York (1999). Examples of amino blockinggroups include, but are not limited to, t-butoxycarbonyl,9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and the like.

The term “protected hydroxyl group,” as used herein, refers to ahydroxyl group protected with a protecting group, as defined above.

The term “acyl” includes residues derived from substituted orunsubstituted acids including, but not limited to, carboxylic acids,carbamic acids, carbonic acids, sulfonic acids, and phosphorous acids.Examples include aliphatic carbonyls, aromatic carbonyls, aliphaticsulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphatesand aliphatic phosphates.

The term “aprotic solvent,” as used herein, refers to a solvent that isrelatively inert to proton activity, i.e., not acting as a proton-donor.Examples include, but are not limited to, hydrocarbons, such as hexaneand toluene, for example, halogenated hydrocarbons, such as, forexample, methylene chloride, ethylene chloride, chloroform, and thelike, heterocyclic compounds, such as, for example, tetrahydrofuran andN-methylpyrrolidinone, and ethers such as diethyl ether,bis-methoxymethyl ether. Such compounds are well known to those skilledin the art, and it will be obvious to those skilled in the art thatindividual solvents or mixtures thereof may be preferred for specificcompounds and reaction conditions, depending upon such factors as thesolubility of reagents, reactivity of reagents and preferred temperatureranges, for example. Further discussions of aprotic solvents may befound in organic chemistry textbooks or in specialized monographs, forexample: Organic Solvents Physical Properties and Methods ofPurification, 4th ed., edited by John A. Riddick et al, Vol. II, in theTechniques of Chemistry Series, John Wiley & Sons, NY, 1986. Aproticsolvents useful in the processes of the present invention includes, butare not limited to, toluene, acetonitrile, DMF, THF, dioxane, MTBE,diethylether, NMP, acetone, hydrocarbons, and haloaliphatics.

The term “protic solvent,” as used herein, refers to a solvent thattends to provide protons, such as an alcohol, for example, methanol,ethanol, propanol, isopropanol, butanol, t-butanol, and the like. Suchsolvents are well known to those skilled in the art, and it will beobvious to those skilled in the art that individual solvents or mixturesthereof may be preferred for specific compounds and reaction conditions,depending upon such factors as the solubility of reagents, reactivity ofreagents and preferred temperature ranges, for example. Furtherdiscussions of protogenic solvents may be found in organic chemistrytextbooks or in specialized monographs, for example: Organic SolventsPhysical Properties and Methods of Purification, 4th ed., edited by JohnA. Riddick et al., Vol. II, in the Techniques of Chemistry Series, JohnWiley & Sons, NY, 1986.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to designate a particularconfiguration unless the text so states; thus a carbon-carbon doublebond or carbon-heteroatom double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion.

Gene Modulation

As used herein, the term “target nucleic acid” or “nucleic acid target”is used for convenience to encompass any nucleic acid capable of beingtargeted including without limitation DNA, RNA (including pre-mRNA andmRNA or portions thereof) transcribed from such DNA, and also cDNAderived from such RNA. In one embodiment of the invention, the targetnucleic acid is a messenger RNA. In another embodiment, the degradationof the targeted messenger RNA is facilitated by a RISC complex that isformed with oligomeric compounds of the invention. In anotherembodiment, the degradation of the targeted messenger RNA is facilitatedby a nuclease such as RNaseH.

The hybridization of an oligomeric compound of this invention with itstarget nucleic acid is generally referred to as “antisense”.Consequently, one mechanism in the practice of some embodiments of theinvention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently suitable to target specificnucleic acid molecules and their functions for such antisenseinhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA.

In the context of the present invention, “modulation” and “modulation ofexpression” mean either an increase (stimulation) or a decrease(inhibition) in the amount or levels of a nucleic acid molecule encodingthe gene, e.g., DNA or RNA. Inhibition is often the desired form ofmodulation of expression and mRNA is often a desired target nucleicacid.

The compounds and methods of the present invention are also useful inthe study, characterization, validation and modulation of smallnon-coding RNAs. These include, but are not limited to, microRNAs(mRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), smalltemporal RNAs (stRNA) and tiny non-coding RNAs (tncRNA) or theirprecursors or processed transcripts or their association with othercellular components.

Small non-coding RNAs have been shown to function in variousdevelopmental and regulatory pathways in a wide range of organisms,including plants, nematodes and mammals. MicroRNAs are small non-codingRNAs that are processed from larger precursors by enzymatic cleavage andinhibit translation of mRNAs. stRNAs, while processed from precursorsmuch like mRNAs, have been shown to be involved in developmental timingregulation. Other non-coding small RNAs are involved in events asdiverse as cellular splicing of transcripts, translation, transport, andchromosome organization.

As modulators of small non-coding RNA function, the compounds of thepresent invention find utility in the control and manipulation ofcellular functions or processes such as regulation of splicing,chromosome packaging or methylation, control of developmental timingevents, increase or decrease of target RNA expression levels dependingon the timing of delivery into the specific biological pathway andtranslational or transcriptional control. In addition, the compounds ofthe present invention can be modified in order to optimize their effectsin certain cellular compartments, such as the cytoplasm, nucleus,nucleolus or mitochondria.

The compounds of the present invention can further be used to identifycomponents of regulatory pathways of RNA processing or metabolism aswell as in screening assays or devices.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound”refers to a polymeric structure capable of hybridizing a region of anucleic acid molecule. This term includes oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics andcombinations of these. Oligomeric compounds are routinely preparedlinearly but can be joined or otherwise prepared to be circular and mayalso include branching. Oligomeric compounds can include double strandedconstructs such as for example two strands hybridized to form doublestranded compounds. The double stranded compounds can be linked orseparate and can include overhangs on the ends. In general, anoligomeric compound comprises a backbone of linked momeric subunitswhere each linked momeric subunit is directly or indirectly attached toa heterocyclic base moiety. Oligomeric compounds may also includemonomeric subunits that are not linked to a heterocyclic base moietythereby providing abasic sites. The linkages joining the monomericsubunits, the sugar moieties or surrogates and the heterocyclic basemoieties can be independently modified giving rise to a plurality ofmotifs for the resulting oligomeric compounds including hemimers,gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond, however, open linear structures aregenerally desired. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside linkagesof the oligonucleotide. The normal internucleoside linkage of RNA andDNA is a 3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA). This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring portions which function ina similar manner to oligonulceotides. Such non-naturally occurringoligonucleotides are often desired over the naturally occurring formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers to asequence of nucleosides that are joined by internucleoside linkages thatdo not have phosphorus atoms. Internucleoside linkages of this typeinclude short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixedheteroatom cycloalkyl, one or more short chain heteroatomic and one ormore short chain heterocyclic. These internucleoside linkages include,but are not limited to, siloxane, sulfide, sulfoxide, sulfone, acetyl,formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl,alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate,sulfonamide, amide and others having mixed N, O, S and CH₂ componentparts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Further included in the present invention are oligomeric compounds suchas antisense oligomeric compounds, antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, alternatesplicers, primers, probes, and other oligomeric compounds whichhybridize to at least a portion of the target nucleic acid. As such,these oligomeric compounds may be introduced in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense oligomeric compoundswhich are “DNA-like” elicit RNAse H. Activation of RNase H, therefore,results in cleavage of the RNA target, thereby greatly enhancing theefficiency of oligonucleotide-mediated inhibition of gene expression.Similar roles have been postulated for other ribonucleases such as thosein the RNase III and ribonuclease L family of enzymes.

While one form of antisense oligomeric compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

In addition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodification so long as these other modifications either alone or incombination with other nucleosides enhance one or more of the desiredproperties described above. Thus, for nucleotides that are incorporatedinto oligonucleotides of the invention, these nucleotides can have sugarportions that correspond to naturally-occurring sugars or modifiedsugars. Representative modified sugars include carbocyclic or acyclicsugars, sugars having substituent groups at one or more of their 2′, 3′or 4′ positions and sugars having substituents in place of one or morehydrogen atoms of the sugar. Additional nucleosides amenable to thepresent invention having altered base moieties and or altered sugarmoieties are disclosed in U.S. Pat. No. 3,687,808 and PCT applicationPCT/US89/02323.

The term “nucleobase,” as used herein, is intended to by synonymous with“nucleic acid base or mimetic thereof.” In general, a nucleobase is anysubstructure that contains one or more atoms or groups of atoms capableof hydrogen bonding to a base of an oligonucleotide.

As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivativesof pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

Modified Nucleobases

Modified nucleobases include, but are not limited to, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Certain of these nucleobases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

The term “universal base” as used herein, refers to a monomer in a firstsequence that can pair with a naturally occuring base, i.e A, C, G, T orU at a corresponding position in a second sequence of a duplex in whichone or more of the following is true: (1) there is essentially nopairing between the two; or (2) the pairing between them occursnon-discriminantly with each of the naturally occurring bases andwithout significant destabilization of the duplex.

For examples of universal bases see Survey and summary: the applicationsof universal DNA base analogs. Loakes, D. Nucleic Acids Research, 2001,29, 12, 2437-2447.

The term “hydrophobic base” as used herein, refers to a monomer in afirst sequence that can pair with a naturally occuring base, i.e A, C,G, T or U at a corresponding position in a second sequence of a duplexin which one or more of the following is true: (1) the hydrophobic baseacts as a non-polar close size and shape mimic (isostere) of one of thenaturally occurring nucleosides; or (2) the hydrophobic base lacks allhydrogen bonding functionality on the Watson-Crick pairing edge.

For examples of adenine isosteres, see Probing the requirements forrecognition and catalysis in Fpg and MutY with nonpolar adenineisosteres. Francis, A W, Helquist, S A, Kool, E T, David, S S. J. Am.Chem. Soc., 2003, 125, 16235-16242 or Structure and base pairingproperties of a replicable nonpolar isostere for deoxyadenosine.Guckian, K M, Morales, J C, Kool, E T. J. Org. Chem., 1998, 63,9652-96565.

For examples of cytosine isosteres, see Hydrolysis of RNA/DNA hybridscontaining nonpolar pyrimidine isostreres defines regions essential forHIV type polypurine tract selection. Rausch, J W, Qu, J, Yi-Brunozzi HY, Kool, E T, LeGrice, S F J. Proc. Natl. Acad. Sci., 2003, 100,11279-11284.

For examples of guanosine isosteres, see A highly effective nonpolarisostere of doeoxguanosine: synthesis, structure, stacking and basepairing. O'Neil, B M, Ratto, J E, Good, K L, Tahmassebi, D C, Helquist,S A, Morales, J C, Kool, E T. J. Org. Chem., 2002, 67, 5869-5875.

For examples of thymidine isosteres, see A thymidine triphosphate shapeanalog lacking Watson-Crick pairing ability is replicated with highsequence selectivity. Moran, S, Ren, R X-F, Kool, E T. Proc. Natl. Acad.Sci., 1997, 94, 10506-10511 or Difluorotoluene, a nonpolar isostere forthymidine, codes specifically and efficiently for adenine in DNAreplication. J. Am. Chem. Soc. 1997, 119, 2056-2057.

The term “promiscuous base” as used herein, refers to a monomer in afirst sequence that can pair with a naturally occuring base, i.e A, C,G, T or U at a corresponding position in a second sequence of a duplexin which the promiscuous base can pair non-discriminantly with more thanone of the naturally occurring bases, i.e. A, C, G, T, or U. For anexample, see Polymerase recognition of syntheticoligodeoxyribonucleotides incorporating degenerate pyrimidine and purinebases. Hill, F.; Loakes, D.; Brown, D. M. Proc. Natl. Acad. Sci., 1998,95, 4258-4263.

The term “size expanded base” as used herein, refers to analogs ofnaturally occurring nuceobases that are larger in size and retain theirWatson-Crick pairing ability. For examples see A four-base pairedgenetic helix with expanded size. Liu, B. Gao, J. Lynch, S R, Saito, D,Maynard, L, Kool, E T., Science, 2003, 302, 868-871 and Toward a newgenetic system with expanded dimension: size expanded analogues ofdeoxyadenosine and thymidine. Liu, H. Goa, J. Maynard, Y. Saito, D,Kool, E T, J. Am. Chem. Soc. 2004, 126, 1102-1109.

The term “fluorinated nucleobase” as used herein, refers to a nucleobaseor nucleobase analog, wherein one or more of the aromatic ringsubstituents is a fluoroine atom. It may be possible that all of thering substituents are fluoroine atoms. For examples of fluorinatednucleobases see fluorinated DNA bases as probes of electrostatic effectsin DNA base stacking. Lai, J S, QU, J, Kool, E T, Angew. Chem. Int. Ed.,2003, 42, 5973-5977 and Selective pairning of polyfluorinated DNA bases,Lai, J S, Kool, E T, J. Am. Chem. Soc., 2004, 126, 3040-3041 and Theeffect of universal fluorinated nucleobases on the catalytic activity ofribozymes, Kloppfer, A E, Engels, J W, Nucleosides, Nucleotides &Nucleic Acids, 2003, 22, 1347-1350 and Synthesis of2′aminoalkyl-substituted fluorinated nucleobases and their influence onthe kinetic properties of hammerhead ribozymes, Klopffer, A E, Engels, JW, ChemBioChem., 2003, 5, 707-716.

In some embodiments of the invention, oligomeric compounds, e.g.oligonucleotides, are prepared having polycyclic heterocyclic compoundsin place of one or more heterocyclic base moieties. A number oftricyclic heterocyclic compounds have been previously reported. Thesecompounds are routinely used in antisense applications to increase thebinding properties of the modified strand to a target strand. The moststudied modifications are targeted to guanosines hence they have beentermed G-clamps or cytidine analogs. Many of these polycyclicheterocyclic compounds have the general formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second oligonucleotide include 1,3-diazaphenoxazine-2-one(R₁₀═O, R₁₁-R₁₄═H) [Kurchavov, et al., Nucleosides and Nucleotides,1997, 16, 1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁-R₁₄═H),[Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀═O, R₁₁=—O—(CH₂)₂—NH₂, R₁₂₋₁₄═H)[Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532].Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification, yet. On the other hand, the gain in helicalstability does not compromise the specificity of the oligonucleotides.The T_(m) data indicate an even greater discrimination between theperfect match and mismatched sequences compared to dC5^(me). It wassuggested that the tethered amino group serves as an additional hydrogenbond donor to interact with the Hoogsteen face, namely the O6, of acomplementary guanine thereby forming 4 hydrogen bonds. This means thatthe increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992,which issued on Dec. 28, 1999, the contents of both are commonlyassigned with this application and are incorporated herein in theirentirety. Such compounds include those having the formula:

-   -   Wherein R₁₁, includes (CH₃)₂N—(CH₂)₂—O—; H₂N—(CH₂)₃—;        Ph-CH₂—O—C(═O)—N(H)—(CH₂)₃—; H₂N—;        Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—;        Phthalimidyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—;        Ph-CH₂—O—C(═O)—N(H)—(CH₂)₂—O—; Ph-CH₂—O—C(═O)—N(H)—(CH₂)₃—O—;        (CH₃)₂N—N(H)—(CH₂)₂—O—; Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₂—O—;        Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—O—; H₂N—(CH₂)₂—O—CH₂—;        N₃—(CH₂)₂—O—CH₂—; H₂N—(CH₂)₂—O—, and NH₂C(═NR)NH—.

Also disclosed are tricyclic heterocyclic compounds of the formula:

wherein:

-   -   R_(10a) is O, S or N—CH₃; R_(11a) is A(Z)_(x1), wherein A is a        spacer and Z independently is a label bonding group optionally        bonded to a detectable label, but R_(11a) is not amine,        protected amine, nitro or cyano; and R_(b) is independently        —CH═, —N═, —C(C₁₋₈ alkyl)═ or —C(halogen)═, but no adjacent        R_(b) are both —N═, or two adjacent R_(b) are taken together to        form a ring having the structure:

where R_(c) is independently —CH═, —N═, —C(C₁₋₈ alkyl)═ or —C(halogen)═,but no adjacent R_(b) are both —N═.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity [Lin, K.-Y.; Matteucci, M.J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement waseven more pronounced in case of G-clamp, as a single substitution wasshown to significantly improve the in vitro potency of a 20 mer2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further tricyclic and tetracyclic heteroaryl compounds amenable to thepresent invention include those having the formulas:

-   -   wherein R₁₄ is NO₂ or both R₁₄ and R₁₂ are independently —CH₃.        The synthesis of these compounds is disclosed in U.S. Pat. No.        5,434,257, which issued on Jul. 18, 1995, U.S. Pat. No.        5,502,177, which issued on Mar. 26, 1996, and U.S. Pat. No.        5,646,269, which issued on Jul. 8, 1997, the contents of which        are commonly assigned with this application and are incorporated        herein in their entirety (hereinafter referred to as the “'257,        '177 and '269 patents”).

Further tricyclic heterocyclic compounds amenable to the presentinvention also disclosed in the '257, '177 and '269 patents includethose having the formula:

wherein a and b are independently 0 or 1 with the total of a and b being0 or 1; A is N, C or CH; Y is S, O, C═O, NH or NCH₂, R⁶; Z is C═O; B istaken together with A to form an aryl or heteroaryl ring structurecomprising 5 or 6 ring atoms wherein the heteroaryl ring comprises asingle O ring heteroatom, a single N ring heteroatom, a single S ringheteroatom, a single O and a single N ring heteroatom separated by acarbon atom, a single S and a single N ring heteroatom separated by a Catom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ringheteroatoms at least 2 of which are separated by a carbon atom, andwherein the aryl or heteroaryl ring carbon atoms are unsubstituted withother than H or at least 1 nonbridging ring carbon atom is substitutedwith R²⁰ or ═O; or Z is taken together with A to form an aryl ringstructure comprising 6 ring atoms wherein the aryl ring carbon atoms areunsubstituted with other than H or at least 1 nonbridging ring carbonatom is substituted with R⁶ or ═O; R⁶ is independently H, C₁₋₆ alkyl,C₂₋₆alkenyl, C₂₋₆ alkynyl, NO₂, N(R³)₂, CN or halo, or an R⁶ is takentogether with an adjacent B group R⁶ to complete a phenyl ring; R²⁰ is,independently, H, C₁₋₆ alkyl, C₂₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl,NO₂, N(R²¹)₂, CN, or halo, or an R²⁰ is taken together with an adjacentR²⁰ to complete a ring containing 5 or 6 ring atoms, and tautomers,solvates and salts thereof; R²¹ is, independently, H or a protectinggroup; R³ is a protecting group or H; and tautomers, solvates and saltsthereof.

More specific examples of bases included in the “257, 177 and 269”Patents are compounds of the formula:

wherein each R₁₆, is, independently, selected from hydrogen and varioussubstituent groups. Further polycyclic base moieties having the formula:

wherein: A₆ is O or S; A₇ is CH₂, N—CH₃, O or S; each A₈ and A₉ ishydrogen or one of A₈ and A₉ is hydrogen and the other of A₈ and A₉ isselected from the group consisting of:

wherein: G is —CN, —OA₁₀, —SA₁₀, —N(H)A₁₀, —ON(H)A₁₀ or —C(═NH)N(H)A₁₀;Q₁ is H, —NHA₁₀, —C(═O)N(H)A₁₀, —C(═S)N(H)A₁₀ or —C(═NH)N(H)A₁₀; each Q₂is, independently, H or Pg; A₁₀ is H, Pg, substituted or unsubstitutedC₁-C₁₀ alkyl, acetyl, benzyl, —(CH₂)_(p3)NH₂, —(CH₂)_(p3)N(H)Pg, a D orL α-amino acid, or a peptide derived from D, L or racemic α-amino acids;Pg is a nitrogen, oxygen or thiol protecting group; each p1 is,independently, from 2 to about 6; p2 is from 1 to about 3; and p3 isfrom 1 to about 4; are disclosed in U.S. patent application Ser. No.09/996,292 filed Nov. 28, 2001, which is commonly owned with the instantapplication, and is herein incorporated by reference.

Some particularly useful oligomeric compounds of the invention containat least one nucleoside having one, two, three, or more aliphaticsubstituents, an RNA cleaving group, a reporter group, an intercalator,a group for improving the pharmacokinetic properties of an oligomericcompound, or a group for improving the pharmacodynamic properties of anoligomeric compound, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferredmodification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE. Representative aminooxy substituentgroups are described in co-owned U.S. patent application Ser. No.09/344,260, filed Jun. 25, 1999, entitled “Aminooxy-FunctionalizedOligomers”; and U.S. patent application Ser. No. 09/370,541, filed Aug.9, 1999, entitled “Aminooxy-Functionalized Oligomers and Methods forMaking Same;” hereby incorporated by reference in their entirety.

Other particularly advantageous 2′-modifications include2′-methoxy(2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions onnucleosides and oligomers, particularly the 3′ position of the sugar onthe 3′ terminal nucleoside or at a 3′-position of a nucleoside that hasa linkage from the 2′-position such as a 2′-5′ linked oligomer and atthe 5′ position of a 5′ terminal nucleoside. Oligomers may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative United States patents that teachthe preparation of such modified sugars structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned, and each of which is herein incorporated by reference,and commonly owned U.S. patent application Ser. No. 08/468,037, filed onJun. 5, 1995, also herein incorporated by reference.

Representative guanidino substituent groups that are shown in formulaare disclosed in co-owned U.S. patent application Ser. No. 09/349,040,entitled “Functionalized Oligomers”, filed Jul. 7, 1999, issue fee paidon Oct. 23, 2002.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Modified Oligonucleotides”, filed Aug.6, 1999, hereby incorporated by reference in its entirety. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. Therespective ends of this linear polymeric structure can be joined to forma circular structure by hybridization or by formation of a covalentbond, however, open linear structures are generally preferred. Withinthe oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide. The normal internucleoside linkage of RNA and DNA is a3′ to 5′ phosphodiester linkage.

The oligomeric compounds in accordance with this invention can comprisefrom about 8 to about 80 nucleobases (i.e., from about 8 to about 80linked nucleosides). One of ordinary skill in the art will appreciatethat the invention embodies oligomeric compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 12to 50 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length,or any range therewithin.

Suitable oligomeric compounds are oligonucleotides from about 12 toabout 50 nucleobases or from about 15 to about 30 nucleobases.

Oligomer and Monomer Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally desired. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside linkage or in conjunctionwith the sugar ring the backbone of the oligonucleotide. The normalinternucleoside linkage that makes up the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage.

Chimeric Oligomeric Compounds

It is not necessary for all positions in an oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within anoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative U.S.patents that teach the preparation of such hybrid structures include,but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;5,652,355; 5,652,356; and 5,700,922, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligomer Mimetics

Another group of oligomeric compounds amenable to the present inventionincludes oligonucleotide mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligomeric compounds whereinonly the furanose ring or both the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.

Another modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage can be preferably a methylene,ethylene (referred to in the art as ENA), or (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 to 10 (Singh etal., Chem. Commun., 1998, 4, 455-456). LNA analogs display very highduplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10C), stability towards 3′-exonucleolytic degradation and good solubilityproperties. The basic structure of LNA showing the bicyclic ring systemis shown below:

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638). The authors have demonstrated that LNAs confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNAs have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Further oligonucleotide mimetics have been prepared to include bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J.Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogshave been oligomerized using the phosphoramidite approach and theresulting oligomeric compounds containing tricyclic nucleoside analogshave shown increased thermal stabilities (Tm's) when hybridized to DNA,RNA and itself. Oligomeric compounds containing bicyclic nucleosideanalogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in abackbone the backbone. This class of olignucleotide mimetic is reportedto have useful physical and biological and pharmacological properties inthe areas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Modified Internucleoside Linkages

Specific examples of antisense oligomeric compounds useful in thisinvention include oligonucleotides containing modified e.g.non-naturally occurring internucleoside linkages. As defined in thisspecification, oligonucleotides having modified internucleoside linkagesinclude internucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage(phosphorothioate) did not significantly interfere with RNAi activity.Based on this observation, it is suggested that certain oligomericcompounds of the invention can also have one or more modifiedinternucleoside linkages. One phosphorus containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage.

Modified oligonucleotide backbones containing a phosphorus atom thereininclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphos-phonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In some embodiments of the invention, oligomeric compounds have one ormore phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Suitableamide internucleoside linkages are disclosed in the above referencedU.S. Pat. No. 5,602,240.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted sugar moieties. Suitable oligomeric compounds comprise asugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-,S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other oligonucleotides comprise a sugar substituent groupselected from: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Onemodification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. Another modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy(—O—CH₃),aminopropoxy(—OCH₂CH₂CH₂NH₂), allyl(—CH₂—CH═CH₂),—O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups maybe in the arabino (up) position or ribo (down) position. Similarmodifications may also be made at other positions on the oligomericcompound, particularly the 3′ position of the sugar on the 3′ terminalnucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′terminal nucleotide. Oligomeric compounds may also have sugar mimeticssuch as cyclobutyl moieties in place of the pentofuranosyl sugar.Representative U.S. patents that teach the preparation of such modifiedsugar structures include, but are not limited to, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792,747; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety. Further representative sugar substituentgroups include groups of formula Ia or Ib:

-   -   wherein:    -   R_(b) is O, S or NH;    -   R_(d) is a single bond, O, S or C(═O);    -   R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),        N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula Ic;

-   -   R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;    -   R_(r) is —R_(x)—R_(y);    -   each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,        C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl,        substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or        unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a        chemical functional group or a conjugate group, wherein the        substituent groups are selected from hydroxyl, amino, alkoxy,        carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,        alkyl, aryl, alkenyl and alkynyl;    -   or optionally, R_(u) and R_(v), together form a phthalimido        moiety with the nitrogen atom to which they are attached;    -   each R_(w) is, independently, substituted or unsubstituted        C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   R_(k) is hydrogen, an amino protecting group or —R_(x)—R_(y);    -   R_(p) is hydrogen, an amino protecting group or —R_(x)—R_(y);    -   R_(x) is a bond or a linking moiety;    -   R_(y) is a chemical functional group, a conjugate group or a        solid support medium;    -   each R_(m) and R_(n) is, independently, H, an amino protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where said acyl is an acid        amide or an ester;    -   or R_(m) and R_(n), together, are an amino protecting group, are        joined in a ring structure that optionally includes an        additional heteroatom selected from N and O or are a chemical        functional group;    -   R_(i) is OR_(z), SR_(Z), or N(R_(z))₂;    -   each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);    -   R_(f), R_(g) and R_(h) comprise a ring system having from about        4 to about 7 carbon atoms or having from about 3 to about 6        carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are        selected from oxygen, nitrogen and sulfur and wherein said ring        system is aliphatic, unsaturated aliphatic, aromatic, or        saturated or unsaturated heterocyclic;    -   R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,        alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to        about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,        N(R_(k))(R_(m))OR_(k), halo, SR_(k) or CN;    -   m_(a) is 1 to about 10;    -   each mb is, independently, 0 or 1;    -   mc is 0 or an integer from 1 to 10;    -   md is an integer from 1 to 10;    -   me is from 0, 1 or 2; and    -   provided that when mc is 0, md is greater than 1.        Representative substituents groups of Formula Ia are disclosed        in U.S. patent application Ser. No. 09/130,973, filed Aug. 7,        1998, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby        incorporated by reference in its entirety.

Representative cyclic substituent groups of Formula Ib are disclosed inU.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998,entitled “RNA Targeted 2′-Oligomeric compounds that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Sugar substituent groups also include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)H)]₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups that are shown in formula Icare disclosed in co-owned U.S. patent application Ser. No. 09/349,040,entitled “Functionalized Oligomers”, filed Jul. 7, 1999, herebyincorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Conjugates

Another substitution that can be appended to the oligomeric compounds ofthe invention involves the linkage of one or more moieties or conjugateswhich enhance the activity, cellular distribution or cellular uptake ofthe resulting oligomeric compounds. In one embodiment such modifiedoligomeric compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Representative conjugate groups are disclosedin International Patent Application PCT/US92/09196, filed Oct. 23, 1992the entire disclosure of which is incorporated herein by reference.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

3′-Endo Modifications

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as illustrated in Scheme 1a, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation. Along similar lines, oligomeric triggers of RNAi responsemight be composed of one or more nucleosides modified in such a way thatconformation is locked into a C3′-endo type conformation, i.e. LockedNucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), andethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modifiednucleosides amenable to the present invention are shown below in SchemeIa. These examples are meant to be representative and not exhaustive.

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligoncleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below.) Nucleosides knownto be inhibitors/substrates for RNA dependent RNA polymerases (forexample HCV NS5B.

In one aspect, the present invention is directed to oligonucleotidesthat are prepared having enhanced properties compared to native RNAagainst nucleic acid targets. A target is identified and anoligonucleotide is selected having an effective length and sequence thatis complementary to a portion of the target sequence. Each nucleoside ofthe selected sequence is scrutinized for possible enhancingmodifications. A preferred modification would be the replacement of oneor more RNA nucleosides with nucleosides that have the same 3′-endoconformational geometry. Such modifications can enhance chemical andnuclease stability relative to native RNA while at the same time beingmuch cheaper and easier to synthesize and/or incorporate into anoligonulceotide. The selected sequence can be further divided intoregions and the nucleosides of each region evaluated for enhancingmodifications that can be the result of a chimeric configuration.Consideration is also given to the 5′ and 3′-termini as there are oftenadvantageous modifications that can be made to one or more of theterminal nucleosides. The oligomeric compounds of the present inventioninclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double stranded sequence or sequences.Further modifications are also considered such as internucleosidelinkages, conjugate groups, substitute sugars or bases, substitution ofone or more nucleosides with nucleoside mimetics and any othermodification that can enhance the selected sequence for its intendedtarget.

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and 04′-endo pucker. This isconsistent with Berger et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as but not limitedto antisense and RNA interference as these mechanisms require thebinding of a synthetic oligonucleotide strand to an RNA target strand.In the case of antisense, effective inhibition of the mRNA requires thatthe antisense DNA have a very high binding affinity with the mRNA.Otherwise the desired interaction between the synthetic oligonucleotidestrand and target mRNA strand will occur infrequently, resulting indecreased efficacyl.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J.Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages ofthe 2′-MOE substitution is the improvement in binding affinity, which isgreater than many similar 2′ modifications such as O-methyl, O-propyl,and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethylsubstituent also have been shown to be antisense inhibitors of geneexpression with promising features for in vivo use (Martin, P., Helv.Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50,168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; andAltmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative toDNA, the oligonucleotides having the 2′-MOE modification displayedimproved RNA affinity and higher nuclease resistance. Chimericoligonucleotides having 2′-MOE substituents in the wing nucleosides andan internal region of deoxy-phosphorothioate nucleotides (also termed agapped oligonucleotide or gapmer) have shown effective reduction in thegrowth of tumors in animal models at low doses. 2′-MOE substitutedoligonucleotides have also shown outstanding promise as antisense agentsin several disease states. One such MOE substituted oligonucleotide ispresently being investigated in clinical trials for the treatment of CMVretinitis.

In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate. Phosphate protecting groupsinclude those described in U.S. Pat. No. 5,760,209, U.S. Pat. No.5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat.No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S.Pat. No. 6,465,628 each of which is expressly incorporated herein byreference in its entirety.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA:Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesisas appropriate. In addition specific protocols for the synthesis ofoligomeric compounds of the invention are illustrated in the examplesbelow.

Oligonucleotides are generally prepared either in solution or on asupport medium, e.g. a solid support medium. In general a first synthon(e.g. a monomer, such as a nucleoside) is first attached to a supportmedium, and the oligonucleotide is then synthesized by sequentiallycoupling monomers to the support-bound synthon. This iterativeelongation eventually results in a final oligomeric compound or otherpolymer such as a polypeptide. Suitable support medium can be soluble orinsoluble, or may possess variable solubility in different solvents toallow the growing support bound polymer to be either in or out ofsolution as desired. Traditional support medium such as solid supportmedia are for the most part insoluble and are routinely placed inreaction vessels while reagents and solvents react with and/or wash thegrowing chain until the oligomer has reached the target length, afterwhich it is cleaved from the support and, if necessary further worked upto produce the final polymeric compound. More recent approaches haveintroduced soluble supports including soluble polymer supports to allowprecipitating and dissolving the iteratively synthesized product atdesired points in the synthesis (Gravert et al., Chem. Rev., 1997, 97,489-510).

The term support medium is intended to include all forms of supportknown to one of ordinary skill in the art for the synthesis ofoligomeric compounds and related compounds such as peptides. Somerepresentative support medium that are amenable to the methods of thepresent invention include but are not limited to the following:controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g.,Alul, et al., Nucleic Acids Research 1991, 19, 1527); silica-containingparticles, such as porous glass beads and silica gel such as that formedby the reaction of trichloro-[3-(4-chloromethyl)phenyl]propylsilane andporous glass beads (see Parr and Grohmann, Angew. Chem. Internal. Ed.1972, 11, 314, sold under the trademark “PORASIL E” by WatersAssociates, Framingham, Mass., USA); the mono ester of1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, TetrahedronLett., 1970, 4503, sold under the trademark “BIOPAK” by WatersAssociates); TENTAGEL (see, e.g., Wright, et al., Tetrahedron Letters1993, 34, 3373); cross-linked styrene/divinylbenzene copolymer beadedmatrix or POROS, a copolymer of polystyrene/divinylbenzene (availablefrom Perceptive Biosystems); soluble support medium, polyethylene glycolPEG's (see Bonora et al., Organic Process Research & Development, 2000,4, 225-231).

Further support medium amenable to the present invention include withoutlimitation PEPS support a polyethylene (PE) film with pendant long-chainpolystyrene (PS) grafts (molecular weight on the order of 10⁶, (seeBerg, et al., J. Am. Chem. Soc., 1989, 111, 8024 and InternationalPatent Application WO 90/02749),). The loading capacity of the film isas high as that of a beaded matrix with the additional flexibility toaccommodate multiple syntheses simultaneously. The PEPS film may befashioned in the form of discrete, labeled sheets, each serving as anindividual compartment. During all the identical steps of the syntheticcycles, the sheets are kept together in a single reaction vessel topermit concurrent preparation of a multitude of peptides at a rate closeto that of a single peptide by conventional methods. Also, experimentswith other geometries of the PEPS polymer such as, for example,non-woven felt, knitted net, sticks or microwellplates have notindicated any limitations of the synthetic efficacy.

Further support medium amenable to the present invention include withoutlimitation particles based upon copolymers of dimethylacrylamidecross-linked with N,N′-bisacryloylethylenediamine, including a knownamount ofN-tertbutoxycarbonyl-beta-alanyl-N,N′-acryloylhexamethylenediamine.Several spacer molecules are typically added via the beta alanyl group,followed thereafter by the amino acid residue subunits. Also, the betaalanyl-containing monomer can be replaced with an acryloyl safcosinemonomer during polymerization to form resin beads. The polymerization isfollowed by reaction of the beads with ethylenediamine to form resinparticles that contain primary amines as the covalently linkedfunctionality. The polyacrylamide-based supports are relatively morehydrophilic than are the polystyrene-based supports and are usually usedwith polar aprotic solvents including dimethylformamide,dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, etal., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, andJ. C. S. Perkin I 538 (1981)).

Further support medium amenable to the present invention include withoutlimitation a composite of a resin and another material that is alsosubstantially inert to the organic synthesis reaction conditionsemployed. One exemplary composite (see Scott, et al., J. Chrom. Sci.,1971, 9, 577) utilizes glass particles coated with a hydrophobic,cross-linked styrene polymer containing reactive chloromethyl groups,and is supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA.Another exemplary composite contains a core of fluorinated ethylenepolymer onto which has been grafted polystyrene (see Kent andMerrifield, Israel J. Chem. 1978, 17, 243 and van Rietschoten inPeptides 1974, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp.113-116). Contiguous solid support media other than PEPS, such as cottonsheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) andhydroxypropylacrylate-coated polypropylene membranes (Daniels, et al.,Tetrahedron Lett. 1989, 4345). Acrylic acid-grafted polyethylene-rodsand 96-microtiter wells to immobilize the growing peptide chains and toperform the compartmentalized synthesis. (Geysen, et al., Proc. Natl.Acad. Sci. USA, 1984, 81, 3998). A “tea bag” containingtraditionally-used polymer beads. (Houghten, Proc. Natl. Acad. Sci. USA,1985, 82, 5131). Simultaneous use of two different supports withdifferent densities (Tregear, Chemistry and Biology of Peptides, J.Meienhofer, ed., Ann Arbor Sci. Publ., Ann Arbor, 1972 pp. 175-178).Combining of reaction vessels via a manifold (Gorman, Anal. Biochem.,1984, 136, 397). Multicolumn solid-phase synthesis (e.g., Krchnak, etal., Int. J. Peptide Protein Res., 1989, 33, 209), and Holm and Meldal,in “Proceedings of the 20th European Peptide Symposium”, G. Jung and E.Bayer, eds., Walter de Gruyter & Co., Berlin, 1989 pp. 208-210).Cellulose paper (Eichler, et al., Collect. Czech. Chem. Commun., 1989,54, 1746). Support mediumted synthesis of peptides have also beenreported (see, Synthetic Peptides: A User's Guide, Gregory A. Grant, Ed.Oxford University Press 1992; U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re-34,069.)

Support bound oligonucleotide synthesis relies on sequential addition ofnucleotides to one end of a growing chain. Typically, a first nucleoside(having protecting groups on any exocyclic amine functionalitiespresent) is attached to an appropriate glass bead support andnucleotides bearing the appropriate activated phosphite moiety, i.e. an“activated phosphorous group” (typically nucleotide phosphoramidites,also bearing appropriate protecting groups) are added stepwise toelongate the growing oligonucleotide. Additional methods for solid-phasesynthesis may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. No.4,725,677 and Re. No. 34,069.

Commercially available equipment routinely used for the support mediumbased synthesis of oligomeric compounds and related compounds is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. Suitable solid phasetechniques, including automated synthesis techniques, are described inF. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach,Oxford University Press, New York (1991).

The term “linking moiety,” as used herein is generally a di-functionalgroup, covalently binds the ultimate 3′-nucleoside (and thus the nascentoligonucleotide) to the solid support medium during synthesis, but whichis cleaved under conditions orthogonal to the conditions under which the5′-protecting group, and if applicable any 2′-protecting group, areremoved. Suitable linking moietys include, but are not limited to, adivalent group such as alkylene, cycloalkylene, arylene, heterocyclyl,heteroarylene, and the other variables are as described above. Exemplaryalkylene linking moietys include, but are not limited to, C₁-C₁₂alkylene (e.g. preferably methylene, ethylene (e.g. ethyl-1,2-ene),propylene (e.g. propyl-1,2-ene, propyl-1,3-ene), butylene, (e.g.butyl-1,4-ene, 2-methylpropyl-1,3-ene), pentylene, hexylene, heptylene,octylene, decylene, dodecylene), etc. Exemplary cycloalkylene groupsinclude C₃-C₁₂ cycloalkylene groups, such as cyclopropylene,cyclobutylene, cyclopentanyl-1,3-ene, cyclohexyl-1,4-ene, etc. Exemplaryarylene linking moietys include, but are not limited to, mono- orbicyclic arylene groups having from 6 to about 14 carbon atoms, e.g.phenyl-1,2-ene, naphthyl-1,6-ene, napthyl-2,7-ene, anthracenyl, etc.Exemplary heterocyclyl groups within the scope of the invention includemono- or bicyclic aryl groups having from about 4 to about 12 carbonatoms and about 1 to about 4 hetero atoms, such as N, O and S, where thecyclic moieties may be partially dehydrogenated. Certain heteroarylgroups that may be mentioned as being within the scope of the inventioninclude: pyrrolidinyl, piperidinyl (e.g. 2,5-piperidinyl,3,5-piperidinyl), piperazinyl, tetrahydrothiophenyl, tetrahydrofuranyl,tetrahydro quinolinyl, tetrahydro isoquinolinyl, tetrahydroquinazolinyl,tetrahydroquinoxalinyl, etc. Exemplary heteroarylene groups includemono- or bicyclic aryl groups having from about 4 to about 12 carbonatoms and about 1 to about 4 hetero atoms, such as N, O and S. Certainheteroaryl groups that may be mentioned as being within the scope of theinvention include: pyridylene (e.g. pyridyl-2,5-ene, pyridyl-3,5-ene),pyrimidinyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,quinazolinyl, quinoxalinyl, etc.

Suitable reagents for introducing the group HOCO-Q-CO include diacids(HO₂C-Q-CO₂H). Particularly suitable diacids include malonic acid (Q ismethylene), succinic acid (Q is 1,2-ethylene), glutaric acid, adipicacid, pimelic acid, and phthalic acid. Other suitable reagents forintroducing HOCO-Q-CO above include diacid anhydrides. Particularlysuitable diacid anhydrides include malonic anhydride, succinicanhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, andphthalic anhydride. Other suitable reagents for introducing HOCO-Q-COinclude diacid esters, diacid halides, etc. One especially preferredreagent for introducing HOCO-Q-CO is succinic anhydride.

The compound of formula may be linked to a support via terminalcarboxylic acid of the HOCO-Q-CO group, via a reactive group on thesupport medium. In some embodiments, the terminal carboxylic acid formsan amide linkage with an amine reagent on the support surface. In otherembodiments, the terminal carboxylic acid forms an ester with an OHgroup on the support medium. In some embodiments, the terminalcarboxylic acid may be replaced with a terminal acid halide, acid ester,acid anhydride, etc. Specific acid halides include carboxylic chlorides,bromides and iodides. Specific esters include methyl, ethyl, and otherC₁-C₁₀ alkyl esters. Specific anhydrides include formyl, acetyl,propanoyl, and other C₁-C₁₀ alkanoyl esters.

The present invention also encompasses the preparation of oligomericcompounds incorporating at least one 2′-O-protected nucleoside into theoligomeric compounds delineated herein. After incorporation andappropriate deprotection the 2′-O-protected nucleoside will be convertedto a ribonucleoside at the position of incorporation. The number andposition of the 2-ribonucleoside units in the final oligomeric compoundcan vary from one at any site or the strategy can be used to prepare upto a full 2′-OH modified oligomeric compound. All 2′-O-protecting groupsamenable to the synthesis of oligomeric compounds are included in thepresent invention. In general, a protected nucleoside is attached to asolid support by for example a succinate linker. Then theoligonucleotide is elongated by repeated cycles of deprotecting the5′-terminal hydroxyl group, coupling of a further nucleoside unit,capping and oxidation (alternatively sulfurization). In a morefrequently used method of synthesis the completed oligonucleotide iscleaved from the solid support with the removal of phosphate protectinggroups and exocyclic amino protecting groups by treatment with anammonia solution. Then a further deprotection step is normally requiredfor the more specialized protecting groups used for the protection of2′-hydroxyl groups which will give the fully deprotectedoligonucleotide.

A large number of 2′-O-protecting groups have been used for thesynthesis of oligoribonucleotides but over the years more effectivegroups have been discovered. The key to an effective 2′-O-protectinggroup is that it is capable of selectively being introduced at the2′-O-position and that it can be removed easily after synthesis withoutthe formation of unwanted side products. The protecting group also needsto be inert to the normal deprotecting, coupling, and capping stepsrequired for oligoribonucleotide synthesis. Some of the protectinggroups used initially for oligoribonucleotide synthesis includedtetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groupsare not compatible with all 5′-O-protecting groups so modified versionswere used with 5′-DMT groups such as1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identifieda number of piperidine derivatives (like Fpmp) that are useful in thesynthesis of oligoribonucleotides including1-[(chloro-4-methyl)phenyl]4′-methoxypiperidin-4-yl (Reese et al.,Tetrahedron Lett., 1986, (27), 2291). Another approach was to replacethe standard 5′-DMT (dimethoxytrityl) group with protecting groups thatwere removed under non-acidic conditions such as levulinyl and9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile2′-protecting groups for oligoribonucleotide synthesis. Another morewidely used protecting group initially used for the synthesis ofoligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al.,Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett.,1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The2′-O-protecting groups can require special reagents for their removalsuch as for example the t-butyldimethylsilyl group is normally removedafter all other cleaving/deprotecting steps by treatment of theoligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups(Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoridelabile and photolabile protecting groups that are removed using moderateconditions. One photolabile group that was examined was the[2-(nitrobenzyl)oxy]methyl (nbm) protecting group (Schwartz et al.,Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examinedincluded a number structurally related formaldehyde acetal-derived,2′-O-protecting groups. Also prepared were a number of relatedprotecting groups for preparing 2′-O-alkylated nucleosidephosphoramidites including 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was preparedto be used orthogonally to the TOM group was2′-O—[(R)-1-(2-nitrophenyl)ethyloxy)methyl] ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acidlabile) and an acid labile 2′-O-protecting group has been reported(Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number ofpossible silyl ethers were examined for 5′-O-protection and a number ofacetals and orthoesters were examined for 2′-O-protection. Theprotection scheme that gave the best results was 5′-O-silyl ether-2′-ACE(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether(DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses amodified phosphoramidite synthesis approach in that some differentreagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis ofoligoribonucleotides the main RNA synthesis strategies that arepresently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O-[(triisopropylsilyl)oxy]methyl(2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. Such an activator would also be amenable to the presentinvention.

The primary groups being used for commercial RNA synthesis are:

-   -   TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;    -   TOM=2′-O-[(triisopropylsilyl)oxy]methyl;    -   DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl        ether-2′-O-bis(2-acetoxyethoxy)methyl    -   FPMP=5′-O-DMT-2′-O-[1 (2-fluorophenyl)₄-methoxypiperidin-4-yl].

All of the aforementioned RNA synthesis strategies are amenable to thepresent invention. Strategies that would be a hybrid of the above e.g.using a 5′-protecting group from one strategy with a 2′-O-protectingfrom another strategy is also amenable to the present invention.

Targets of the Invention

“Targeting” an antisense oligomeric compound to a particular nucleicacid molecule, in the context of this invention, can be a multistepprocess. The process usually begins with the identification of a targetnucleic acid whose function is to be modulated. This target nucleic acidmay be, for example, a cellular gene (or mRNA transcribed from the gene)whose expression is associated with a particular disorder or diseasestate, or a nucleic acid molecule from an infectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid. The terms region,segment, and site can also be used to describe an oligomeric compound ofthe invention such as for example a gapped oligomeric compound having 3separate segments.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding a nucleic acid target, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense oligomeric compounds of thepresent invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, oneregion is the intragenic region encompassing the translation initiationor termination codon of the open reading frame (ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsosuitable to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also suitable target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense oligomeric compounds targeted to,for example, DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequences.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso suitable target nucleic acids.

The locations on the target nucleic acid to which the antisenseoligomeric compounds hybridize are hereinbelow referred to as “suitabletarget segments.” As used herein the term “suitable target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense oligomeric compound is targeted. While not wishingto be bound by theory, it is presently believed that these targetsegments represent portions of the target nucleic acid which areaccessible for hybridization.

Exemplary antisense oligomeric compounds include oligomeric compoundsthat comprise at least the 8 consecutive nucleobases from the5′-terminus of a targeted nucleic acid e.g. a cellular gene or mRNAtranscribed from the gene (the remaining nucleobases being a consecutivestretch of the same oligonucleotide beginning immediately upstream ofthe 5′-terminus of the antisense compound which is specificallyhybridizable to the target nucleic acid and continuing until theoligonucleotide contains from about 8 to about 80 nucleobases).Similarly, antisense oligomeric compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds (the remaining nucleobases being a consecutive stretch of thesame oligonucleotide beginning immediately downstream of the 3′-terminusof the antisense compound which is specifically hybridizable to thetarget nucleic acid and continuing until the oligonucleotide containsfrom about 8 to about 80 nucleobases). One having skill in the art armedwith the antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further antisense compounds.

Once one or more target regions, segments or sites have been identified,antisense oligomeric compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

In accordance with one embodiment of the present invention, a series ofnucleic acid duplexes comprising the antisense oligomeric compounds ofthe present invention and their complements can be designed for aspecific target or targets. The ends of the strands may be modified bythe addition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the duplex is then designed andsynthesized as the complement of the antisense strand and may alsocontain modifications or additions to either terminus. For example, inone embodiment, both strands of the duplex would be complementary overthe central nucleobases, each having overhangs at one or both termini.

For example, a duplex comprising an antisense oligomeric compound havingthe sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:1) and having atwo-nucleobase overhang of deoxythymidine(dT) would have the followingstructure:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from various RNA synthesis companies such as for exampleDharmacon Research Inc., (Lafayette, Colo.). Once synthesized, thecomplementary strands are annealed. The single strands are aliquoted anddiluted to a concentration of 50 uM. Once diluted, 30 uL of each strandis combined with 15 uL of a 5× solution of annealing buffer. The finalconcentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. Thissolution is incubated for 1 minute at 90° C. and then centrifuged for 15seconds. The tube is allowed to sit for 1 hour at 37° C. at which timethe dsRNA duplexes are used in experimentation. The final concentrationof the dsRNA compound is 20 uM. This solution can be stored frozen (−20°C.) and freeze-thawed up to 5 times.

Once prepared, the desired synthetic duplexs are evaluated for theirability to modulate target expression. When cells reach 80% confluency,they are treated with synthetic duplexs comprising at least oneoligomeric compound of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired dsRNA compound at a finalconcentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

In a further embodiment, the “suitable target segments” identifiedherein may be employed in a screen for additional oligomeric compoundsthat modulate the expression of a target. “Modulators” are thoseoligomeric compounds that decrease or increase the expression of anucleic acid molecule encoding a target and which comprise at least an8-nucleobase portion which is complementary to a suitable targetsegment. The screening method comprises the steps of contacting asuitable target segment of a nucleic acid molecule encoding a targetwith one or more candidate modulators, and selecting for one or morecandidate modulators which decrease or increase the expression of anucleic acid molecule encoding a target. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding a target, the modulator may then be employed in furtherinvestigative studies of the function of a target, or for use as aresearch, diagnostic, or therapeutic agent in accordance with thepresent invention.

The suitable target segments of the present invention may also becombined with their respective complementary antisense oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides.

Hybridization

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,one mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An antisense oligomeric compound is specifically hybridizable whenbinding of the compound to the target nucleic acid interferes with thenormal function of the target nucleic acid to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense oligomeric compound to non-targetnucleic acid sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and under conditions in which assaysare performed in the case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention, “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases regardless of where the two are located. Forexample, if a nucleobase at a certain position of an oligomeric compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, the target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleobases which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleobases such that stable and specificbinding occurs between the oligonucleotide and a target nucleic acid.

It is understood in the art that the sequence of an antisense oligomericcompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). The antisense oligomeric compounds ofthe present invention can comprise at least about 70%, at least about80%, at least about 90%, at least about 95%, or at least about 99%sequence complementarity to a target region within the target nucleicacid sequence to which they are targeted. For example, an antisenseoligomeric compound in which 18 of 20 nucleobases of the antisenseoligomeric compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense oligomeric compound which is 18nucleobases in length having 4 (four) noncomplementary nucleobases whichare flanked by two regions of complete complementarity with the targetnucleic acid would have 77.8% overall complementarity with the targetnucleic acid and would thus fall within the scope of the presentinvention. Percent complementarity of an antisense oligomeric compoundwith a region of a target nucleic acid can be determined routinely usingBLAST programs (basic local alignment search tools) and PowerBLASTprograms known in the art (Altschul et al., J. Mol. Biol., 1990, 215,403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

Screening and Target Validation

In some embodiments, “suitable target segments” may be employed in ascreen for additional oligomeric compounds that modulate the expressionof a selected protein. “Modulators” are those oligomeric compounds thatdecrease or increase the expression of a nucleic acid molecule encodinga protein and which comprise at least an 8-nucleobase portion which iscomplementary to a suitable target segment. The screening methodcomprises the steps of contacting a suitable target segment of a nucleicacid molecule encoding a protein with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding a protein.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a peptide, the modulator may then beemployed in further investigative studies of the function of thepeptide, or for use as a research, diagnostic, or therapeutic agent inaccordance with the present invention.

The suitable target segments of the present invention may also becombined with their respective complementary antisense oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides. Such double stranded oligonucleotidemoieties have been shown in the art to modulate target expression andregulate translation as well as RNA processsing via an antisensemechanism. Moreover, the double-stranded moieties may be subject tochemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmonsand Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al.,Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., GenesDev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498;Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target (Tijsterman etal., Science, 2002, 295, 694-697).

The oligomeric compounds of the present invention can also be applied inthe areas of drug discovery and target validation. The present inventioncomprehends the use of the oligomeric compounds and targets identifiedherein in drug discovery efforts to elucidate relationships that existbetween proteins and a disease state, phenotype, or condition. Thesemethods include detecting or modulating a target peptide comprisingcontacting a sample, tissue, cell, or organism with the oligomericcompounds of the present invention, measuring the nucleic acid orprotein level of the target and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furtheroligomeric compound of the invention. These methods can also beperformed in parallel or in combination with other experiments todetermine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature (2001), 411,494-498; Nishikura et al., Cell (2001), 107, 415-416; and Bass et al.,Cell (2000), 101, 235-238.)

Kits, Research Reagents, Diagnostics, and Therapeutics

The oligomeric compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the oligomeric compounds of the presentinvention, either alone or in combination with other oligomericcompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense oligomeric compounds are compared tocontrol cells or tissues not treated with antisense oligomeric compoundsand the patterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds and or oligomeric compounds which affectexpression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The oligomeric compounds of the invention are useful for research anddiagnostics, because these oligomeric compounds hybridize to nucleicacids encoding proteins. For example, oligonucleotides that are shown tohybridize with such efficiency and under such conditions as disclosedherein as to be effective protein inhibitors will also be effectiveprimers or probes under conditions favoring gene amplification ordetection, respectively. These primers and probes are useful in methodsrequiring the specific detection of nucleic acid molecules encodingproteins and in the amplification of the nucleic acid molecules fordetection or for use in further studies. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid can be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofselected proteins in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligomeric compoundshave been employed as therapeutic moieties in the treatment of diseasestates in animals, including humans. Antisense oligonucleotide drugs,including ribozymes, have been safely and effectively administered tohumans and numerous clinical trials are presently underway. It is thusestablished that antisense oligomeric compounds can be usefultherapeutic modalities that can be configured to be useful in treatmentregimes for the treatment of cells, tissues and animals, especiallyhumans.

For therapeutics, an animal, such as a human, suspected of having adisease or disorder which can be treated by modulating the expression ofa selected protein is treated by administering antisense oligomericcompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a protein inhibitor. The protein inhibitors of the present inventioneffectively inhibit the activity of the protein or inhibit theexpression of the protein. In some embodiments, the activity orexpression of a protein in an animal or cell is inhibited by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 99%,or by 100%.

For example, the reduction of the expression of a protein may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. The cells contained within the fluids, tissuesor organs being analyzed can contain a nucleic acid molecule encoding aprotein and/or the protein itself.

The oligomeric compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of anoligomeric compound to a suitable pharmaceutically acceptable diluent orcarrier. Use of the oligomeric compounds and methods of the inventionmay also be useful prophylactically.

In another embodiment, the present invention provides for the use of acompound(s) of the invention in the manufacture of a medicament for thetreatment of any and all diseases and conditions disclosed herein.

Formulations

The antisense oligomeric compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the oligomeric compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligomeric compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

While the present invention has been described with specificity inaccordance with certain of its embodiments, the following examples serveonly to illustrate the invention and are not intended to limit the same.

Synthetic Schemes

The compounds and processes of the present invention will be betterunderstood in connection with the following synthetic schemes which areillustrative of the methods by which the compounds of the invention maybe prepared.

EXAMPLES

The compounds and processes of the present invention will be describedfurther in detail with respect to specific preferred embodiments by wayof examples, it being understood that these are intended to beillustrative only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1 1-O-methyl-D-ribose (2)

D-ribose (500 g, 3.33 moles) was dissolved in 1.5 L of dry methanolcontaining 0.5% hydrochloric acid and stirred in a flask equipped with acalcium carbonate drying tube for 48 hrs. The reaction mixture wasneutralized with Dowex (OH form) resin to pH 7. The reaction mixture wasfiltered and concentrated under reduced pressure followed by drying invacuo overnight to give compound 2 (550 g) in 98% yield.

Example 2 2,3,5-tri-O-benzyl-1-O-methyl-D-ribose (3)

Compound 2 (150 g, 0.914 mole) was dissolved in dryN,N-dimethylformamide (DMF, 1.5 L), under nitrogen, in a three neckedflask equipped with a mechanical stirrer and an addition funnel. Thesolution was cooled to 0° C. in an ice bath. NaH (220 g, 60% dispersionin mineral oil, 5.48 mole, 6 eq.) was added in small portions takingcare to control the reaction and avoid overheating. When all the NaH hadbeen added, addition of benzyl bromide (650 mL, 5.48 mole, 6 eq.) wasinitiated drop-wise via the addition funnel. When all the benzyl bromidehad been added the reaction was allowed to come to room temperature andstirred for 5 hrs. The reaction was then heated to 60° C. and stirred atthis temperature overnight. The reaction was quenched with methanol,concentrated in vacuo, and partitioned between ether and water. Theether layer was washed once with 10% citric acid solution, once withsaturated sodium bicarbonate solution and once with brine. The etherlayer was dried over anhydrous sodium sulfate, and concentrated to apale, yellow syrup. This syrup was dissolved in 5% ethyl acetate inhexane and applied to a silica gel plug in a 2 L sintered glass funnel.5% ethyl acetate in hexane was used to elute the desired product in 2 Lfractions. The target fractions were concentrated under reduced pressureto give compound 3 (300 g) in 75% yield.

Example 3 2,3,5-Tri-O-benzyl-D-ribitol (4)

Compound 3 (300 g, 0.688 mole) was treated as per procedures describedin Naka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtaincompound 4 (200 g) in 70% yield.

Example 4 2,3,5-Tri-O-benzyl-1-O-tert-butyldiphenylsilyl-D-ribitol (5)

To an ice-cold solution of Compound 4 (200 g, 0.47 mole) in dry pyridine(1 L), under nitrogen, was added tert-butyldiphenylsilyl chloride (130mL, 0.50 mole, 1.05 eq.) slowly with vigorous stirring. The reactionmixture was allowed to come to room temperature and stirred overnight.The reaction was then quenched with methanol, and solvent was removedunder reduced pressure. The residue was taken up in ethyl acetate andwashed successively with, water (twice), 10% citric acid solution, sat.sodium bicarbonate solution, and brine. The ethyl acetate layer wasseparated, dried over anhydrous sodium sulfate, and concentrated underreduced pressure to give compound 5 (400 g) in 98% yield. The materialwas of sufficient purity to be used without further purification.

Example 52,3,5-Tri-O-benzyl-1-O-tert-butyldiphenylsilyl-4-O-p-nitrobenzoyl-L-lyxitol(6)

Compound 5 (150 g, 232.5 mmole) was treated as per procedures describedin Naka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtainCompound 6 (130 g) in 70% yield.

Example 6 2,3,5-Tri-O-benzyl-L-lyxitol (7)

Compound 6 (110 g, 138.54 mmole) was dissolved in methanol (500 mL) andsodium methoxide (11 g, 204.0 mmole, 1.5 eq.) was added and the solutionwas stirred at room temperature for 2 hours. The reaction mixture wasconcentrated under reduced pressure, and the residue was partitionedbetween ether and water. The separated organic layer was washed withbrine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was dried in vacuo for 3 hrs and dissolvedin dry THF under nitrogen. Triethylamine (TEA) (100 mL, 0.752 mole, 5eq.) was added followed by triethylamine trihydrofluoride (TREAT.HF)(220 mL, 1.38 mole, 10 eq.) and the mixture was stirred overnight atroom temperature. The solvent was removed under reduced pressure and theresidue partitioned between ether and water. The ether layer was washedwith brine and dried over anhydrous sodium sulfate. The solvent wasremoved under reduced pressure and the residue purified by flashchromatography (40% ethyl acetate in hexane) to give 51 g of compound 7in 90% yield (reported literature yield 81%).

Example 7 1,4-anhydro-2,3,5-tri-O-benzyl-4-thio-D-ribitol (8)

Compound 7 (51 g, 121.0 mmole) was treated as per procedures describedin Naka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtain 47g of 8 in 85% yield.

Example 8 1,4-anhydro-4-thio-D-ribitol (9)

Compound 8 (47 g, 112 mmole) was treated as per procedures described inNaka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtainCompound 9 (13 g) in 80% yield.

Example 91,4-anhydro-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-D-ribitol(10)

Compound 9 (15 g, 100.0 mmole) was treated as per procedures describedin Naka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtainCompound 10 (24 g) in 80% yield.

Example 101,4-anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-D-ribitol(11)

Compound 10 (24 g, 61 mmole) was treated as per procedures described inNaka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtainCompound 11 (30 g) in 90% yield.

Example 111,4-anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-sulfinyl-D-ribitol (11a)

The title compound was prepared by a new route (followed similarprecedence, to synthesize asymmetric 1,3-Dithiolane Nucleoside analogs,Romualo Caputo et. al. Eur. J. Org. Chem. 2003, 346-350). To a solutionof Ti(IV) isopropoxide (0.3 mL, 1.10 mmole, 0.45 eq.) in drydichloromethane (5 mL), under nitrogen was added diethyl-L-tartrate (0.6mL, 1.15 mmole, 1.5 eq) with vigorous stirring. The stirring wascontinued for 20 min at room temperature until a clear straw yellowcolored solution was obtained. This solution was cooled to −15° C. to−20° C. and tert-butyl hydroperoxide was added (0.4 mL of 6 M solutionin decane, 2.4 mmole). The solution was stirred at the same temperaturefor 5 minutes and a solution of Compound 10 (1.3 g, 2.3 mmole) in drydichloromethane (5 mL) was added. The reaction mixture was stirred atthe same temperature for 24 hrs when TLC indicated that all startingmaterial had been consumed. The reaction was quenched by the addition ofwater and allowed to come to room temperature. The reaction mixture wastransferred to a separating funnel and partitioned between brine anddichloromethane. The brine layer was extracted several times withdichloromethane and the combined dichloromethane extracts were driedover anhydrous sodium sulfate. The cloudy solution was clarified byfiltration through a celite pad and concentrated to syrup under reducedpressure. The residue was purified by flash chromatography using 5 to10% ethyl acetate in dichloromethane as eluant to obtain Compound 11(1.2 g) in 90% yield. ¹H NMR indicated that the product contained onlythe desired (R) isomer that corresponded to the literature values. Theproduct was contaminated with residual L-diethyl-tartrate but since thisimpurity does not interfere with the subsequent steps the material wasused as such without further purification.

Example 121-[2-O-(2,4,-dimethoxybenzoyl)-3,5-O-1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]uracil(12)

Compound 11 (1.2 g, 2.1 mmole) was treated as per procedures describedin Naka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtainCompound 12 (1.3 g) in 90% yield.

Example 13 1-(4-thio-β-D-ribofuranosyl)uracil (13)

Compound 12 (1.2 g, 1.90 mmole) was dissolved in dry THF under nitrogen.TEA (1.3 mL, 9.5 mmole, 5 eq.) and TREAT.HF (2.8 mL, 20 mmole, 10 eq.)were added and the mixture stirred at room temperature for 4 to 5 h. Thesolvent was removed under reduced pressure and the residue wasco-evaporated three times with toluene. Methanolic ammonia (20 mL) wasadded and the reaction mixture was stirred at room temperatureovernight. The solvent was removed under reduced pressure and theresidue was purified by flash chromatography (10% methanol indichloromethane) to give Compound 13 (420 mg) in 90% yield.

Compound 13 was also synthesized using literature procedure (Naka et al.in J. Am. Chem. Soc., Vol. 122, No. 30, 2000) with few modificationsthat resulted in significant increase in yields.

Example 14 1-[5-(4,4′-dimethoxytrityl)-4-thio-β-D-ribofuranosyl]uracil(14)

Compound 13 (2 g, 7.754 mmol) was dissolved in dry pyridine (20 mL),4,4′-dimethoxytrityl chloride (2.9 g, 8.53 mmol) was added and themixture stirred at room temperature overnight. The reaction was quenchedwith methanol and taken up in ethyl acetate (200 mL). The ethyl acetatesolution was washed twice with saturated sodium bicarbonate solution,once with water, once with brine, and dried over anhydrous sodiumsulfate. The solvents were removed under reduced pressure and theresidue purified by flash chromatography (7% methanol indichloromethane). The desired fractions were pooled and concentrated togive Compound 14 (3.0 g) in 80% yield. The structure of Compound 14 wasconfirmed by ¹H NMR & ESMS.

Example 151-[5-(4,4′-dimethoxytrityl)-2-(t-butyldimethylsilyl)-4-thio-β-D-ribofuranosyl]uracil(15a-c)

Compound 14 (4.3 g, 7.66 mmol), silver nitrate (3.1 g, 18.5 mmol),anhydrous pyridine (4.5 mL, 56.0 mmol) and TBDMS-Cl (2.8 g, 18.57 mmol)were dissolved in dry THF (75 mL) and stirred at room temperatureovernight. The precipitated silver chloride was removed by filtrationthrough a pad of celite and washed with several portions of THF. Thecombined washings were concentrated to furnish a foam, which waspurified by flash chromatography (40% ethyl acetate in hexane) to giveCompound 15a (3.2 g, 63%), 15b (1.2 g, 23%) & 15c (0.43 g, 8.3%). Thestructures were confirmed by ¹H NMR.

Example 161-(3-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-5-O-(4,4′-dimethoxytrityl)-2-O-tert.Butyldimethylsilyl)-4-thio-β-D-ribofuranosyl)uracil (16)

To a solution of Compound 15a (2.5 g, 3.7 mmol) in anhydrous DMF (13 mL)was added dried 1H-tetrazole (0.2 g, 2.8 mmol), N-methylimidazole (0.077g, 0.9 mmol) and 2-cyanoethoxy-N,N,N′,N′-tetraisopropylphosphoramidite(1.6 g, 5.30 mmol). The reaction mixture was stirred at room temperaturefor 8 h. It was taken up in EtOAc (200 mL) and washed with brine (5×50mL). The organic layer was concentrated and the resulting oil purifiedby column chromatography using 10% acetone in dichloromethane as theeluent. Appropriate fractions were collected and concentrated to a foam,which was dried for two days under high vacuum to furnish pure amidite,yield, 2.96 g, 91.6%. 1H and 31P NMR indicated the correct structure ofCompound 16.

Example 17N⁴-Acetyl-1-[2-O-(2,4,-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]cytosine(17)

Compound 11 (1.2 g, 2.1 mmole) is treated as per procedures described inNaka et al. in J. Am. Chem. Soc., Vol. 122, No. 30, 2000 to obtainCompound 17.

Example 18 1-(4-thio-β-D-ribofuranosyl)cytosine (18)

Compound 17 (1 eq.) is dissolved in dry THF under nitrogen. TEA (1.3 mL,9.5 mmole, 5 eq.) and TREAT.HF (2.8 mL, 20 mmole, 10 eq.) are added andthe mixture stirred at room temperature for 4 to 5 h. The solvent isremoved under reduced pressure and the residue is co-evaporated threetimes with toluene. Methanolic ammonia (2 mL) is added and the reactionmixture is stirred at room temperature overnight. The solvent is removedand the residue is purified by flash chromatography (10% methanol inCH₂Cl₂ containing 0.1% TEA) to give Compound 18.

Example 191-[2,3-di-O-acetyl-5-(4,4′-dimethoxytrityl)-4-thio-β-D-ribofuranosyl]uracil(19)

Compound 14 (3.0 g, 5.36 mmol) was dissolved in dry pyridine (25 mL).Acetic anhydride (5 mL, 53.6 mmol) was added and the mixture was stirredat room temperature for 16 hrs. The reaction was quenched with methanoland taken up in ethyl acetate (100 mL). The ethyl acetate solution waswashed twice with saturated sodium bicarbonate solution, once withwater, once with brine, and dried over anhydrous sodium sulfate. Thesolvents were removed under reduced pressure and the residue purified byflash chromatography (2% methanol in dichloromethane). The desiredfractions were pooled and concentrated to give Compound 19 (2.5 g) in75% yield.

Example 20 1-[5-(4,4′-dimethoxytrityl)-4-thio-β-D-ribofuranosyl]cytosine(20)

1,2,4-triazole (3.73 g, 54 mmol) was suspended in dry acetonitrile (42mL) and cooled to 0° C. in an ice bath. POCl₃ (1.44 mL, 15.4 mmol) wasadded drop wise with stirring. TEA (10.73 mL, 77 mmol) was added slowlywith vigorous stirring and the mixture was stirred at this temperaturefor an additional 30 min. A solution of Compound 19 (2.43 g, 3.85 mmol)in dry acetonitrile (14 mL) was added and the mixture stirred at 0° C.for 2 h and at room temperature for 1 hr. The solvents were removedunder reduced pressure and the residue taken up in ethyl acetate. Theethyl acetate solution was washed twice with saturated sodiumbicarbonate solution, once with brine and dried over anhydrous sodiumsulfate. The solvents were removed under reduced pressure and theresidue was taken up in dioxane (40 mL) and 30% ammonium hydroxidesolution (20 mL) was added. The mixture was stirred in a sealed flaskovernight at room temperature. The solvents were removed under reducedpressure and the residue purified by flash chromatography (10% methanolin dichloromethane) to give Compound 20, (2.0 g) in 75% yield.

Example 21N-benzoyl-1-[5-(4,4′-dimethoxytrityl)-4-thio-β-D-ribofuranosyl]cytosine(21)

Compound 20 (2.0 g, 3.6 mmole) was dissolved in dry DMF (20 mL) andbenzoic anhydride (1.63 g, 7.2 mmol) was added. The mixture was stirredat room temperature overnight. The reaction was quenched by pouring intoice cold saturated sodium bicarbonate solution and stirred for 30 min.The mixture was transferred to a separating funnel and extracted threetimes with dichloromethane. The organic layer was dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by flash chromatography (5% methanol in dichloromethane) togive Compound 21 (2.0 g) in 85% yield.

Example 22N-benzoyl-1-[5-(4,4′-dimethoxytrityl)-2-(t-butyldimethylsilyl)-4-thio-β-D-ribofuranosyl]cytosine(23)

Compound 21 (200 mg, 0.3 mmol), silver nitrate (100 mg, 0.6 mmol), andTBDMS-Cl (70 mg, 0.45 mmol) were dissolved in dry THF and stirred atroom temperature overnight. The reaction was diluted with ethyl acetateand washed with saturated sodium bicarbonate solution, water and brine.The organic layer was dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by flashchromatography (30% ethyl acetate in hexanes) to give Compound 23 (100mg) in 45% yield. The 3-protected analog(N-benzoyl-1-[5-(4,4′-dimethoxytrityl)-3-(t-butyldimethylsilyl)-4-thio-β-D-ribofuranosyl]cytosine)was also isolated as a minor product.

Example 23N⁴-Bz-1-(3-O-2-Cyanoethoxy(diisopropylamino)phosphino)-5-O-(4,4′-dimethoxy-trityl)-2-O-tert.Butyldimethylsilyl)-4-thio-β-D-ribofuranosyl)cytosine (24)

Compound 23 (0.49 g, 0.628 mmol) in anhydrous DMF (3 mL) was added dried1H-tetrazole (0.04 g, 0.56 mmol), N-methylimidazole (0.013 g) and2-cyanoethoxy-N,N,N′,N′-tetraisopropylphosphoramidite (0.286 g, 0.948mmol). The reaction mixture was stirred at room temperature for 8 h andthen taken up in EtOAc (20 mL) and washed with brine (5×10 mL). Theorganic layer was dried over anhydrous sodium sulfate and concentrated.The resulting oil was purified by column chromatography using 10%acetone in dichloromethane as the eluent. Appropriate fractions werecollected and concentrated to a foam, which was dried for two days underhigh vacuum to furnish pure amidite, yield, 0.552 mg, 89%. 1H and 31PNMR indicated the correct structure of compound 24.

Example 24 Synthesis of 4′-thioadenosine

The synthesis of this compound is carried out according to theliterature procedure by Naka et al. in J. Am. Chem. Soc., Vol. 122, No.30, 2000.

Example 25N6-benzoyl-5′-dimethoxytrityl-2′-O-tert-butyldimethylsilyl-3′-O-(cyanoethoxy-N,Ndiisopropyl-phophoramidite)-4′-thio-adenosine

The title compound is prepared from 4′-thioadenosine, using the standardprocedures (Claudine Leydier et.al., Antisense Research and Development5, 1995, 167 and the references therein & Claudine Leydier et.al.Nucleosides & Nucleotides, 13, 1994, 2035 and the references therein.)

Example 26 Synthesis of 4′-Thioguanosine (33)

The title compound is prepared according to literature proceduresstarting with Compound 11 (Naka et al. in J. Am. Chem. Soc., Vol. 122,No. 30, 2000.)

Example 27N²-isobutyryl-5′-dimethoxytrityl-2′-O-tert-butyldimethylsilyl-3′-O-(cyanoethoxy-N,N-diisopropyl-phophoramidite)-4′-thio-guanosine(34)

This compound is prepared from 4′-thioguanosine using one of a number ofliterature procedures (Masad J. Damha & Kelvin K. Ogilvie in ““Methodsin Molecular Biology, Vol. 20: page 81 (and the references therein)Protocols for Oligonucleotide and Analogs; Edited by: S. Agarwal HumanaPress Inc, Totowa, N.J. Oligonucleotide synthesis a practical approach.(1984) M. J. Gait editor IRL Press, Oxford; Scaringe, Stephen A.;Francklyn, Christopher; Usman, Nassim, Chemical synthesis ofbiologically active oligoribonucleotides using b-cyanoethyl protectedribonucleoside phosphoramidites, Nucleic Acids Research (1990), 18(18),5433-41.)

Example 28 Synthesis of 2′-OMe-4′-thio-U Phosphoramidite

1-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]uracil(35)

Compound 13 (1.1 g, 4.23 mmole) was dissolved in anhydrous pyridine (10mL) under a nitrogen atmosphere and cooled in an ice bath. TIPDSCl₂ (1.4mL, 4.44 mmole) was added drop-wise with vigorous stirring. Stirring wascontinued at the same temperature for an additional 2 to 4 h. When allof 13 had been consumed the reaction was quenched by pouring onto ice.The mixture was separated between ethyl acetate and water and the ethylacetate layer was washed thrice with cold sat. sodium bicarbonatesolution and once with brine. The ethyl acetate layer was dried overanhydrous sodium sulfate and concentrated under reduced pressure. Thecrude material was purified by flash chromatography (5% methanol indichloromethane) to give Compound 35 (1.7 g, 80% yield).

3-N-benzoyl-1-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]uracil(36)

A mixture of Compound 35 (250 mg, 0.5 mmole), Na₂CO₃ (424 mg, 4 mmole)and tetrabutylammonium bromide (7 mg, 0.02 mmole) were dissolved in abiphasic mixture of CH₂Cl₂—H₂O. Benzoyl chloride (87 μL, 0.75 mmole) wasadded and the mixture was stirred at room temperature until all ofCompound 35 was consumed. The mixture was transferred to a separatingfunnel. The organic phase was collected and the aqueous phase extractedtwice with CH₂Cl₂. The organic extracts were combined, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was taken up in 1,2-dichloroethane and heated at 60° C. for 15min. The solvent was removed under reduced pressure and the residuepurified by flash chromatography (10% acetone in dichloromethane) toobtain Compound 36 (225 mg, 75% yield).

3-N-benzoyl-1-[2-O-methyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]uracil(37)

Compound 36 (100 mg, 0.165 mmole) was dissolved in dry DMF under aninert atmosphere. Silver oxide (383 mg, 1.65 mmole) and iodomethane (200μL, 3.3 mmole) were added and the mixture stirred at room temperatureovernight. Methanol was added to quench the reaction and the reactionmixture was partitioned between ethyl acetate and water. The ethylacetate layer was washed twice with water, once with brine, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by flash chromatography (gradient ethyl acetate inhexanes) to give Compound 37 (67 mg, 65% yield).

1-(2-O-methyl-4-thio-AD-ribofuranosyl)uracil (38)

Compound 37 (260 mg, 0.42 mmole) was dissolved in dry THF (10 mL).TREAT.HF (1.5 mL) and TEA (0.75 mL) were added and the mixture stirredfor 4 hrs. The solvent was removed under reduced pressure followed bytwo co-evaporations with dry toluene. The residue was taken up inmethanolic ammonia (5 mL) and stirred in a sealed tube overnight. Thesolvents were removed under reduced pressure and the residue purified byflash chromatography (10% methanol in dichloromethane) to give Compound38 (100 mg, 85% yield). ¹H NMR (DMSO-d₆) δ 11.35 (br s, 1H), 8.0 (d,1H), 5.97 (d, 1H), 5.64 (d, 1H), 5.2 (br s, 2H), 4.2 (d, 1H), 3.9 (d,1H), 3.58 (dd, 2H), 3.26 (s, 3H), 1.18 (m, 1H).

1-(5-(4,4′-Dimethoxy-trityl)-2-O-methyl-4-thio-β-D-ribofuranosyl)uracil(39)

Compound 38 (400 mg, 1.46 mmole) was dissolved in dry pyridine in cold.Dimethoxytrityl chloride (600 mg, 1.752 mmole) was added and the mixturewas stirred at room temperature for 24 hrs. Solvents were removed underreduced pressure and the residue was purified by flash chromatography(1:1 ethyl acetate: dichloromethane) to give Compound 39 (600 mg, 75%yield).

2′-O-methyl-4′-thio-U Phosphoramidite (40)

Compound 39 is converted into its corresponding phosphoramidite Compound107 according to standard procedures. (Oligonucleotide Synthesis: APractical Approach. Gait, M. J. (Editor) UK. (1984), Publisher: (IRLPress, Oxford, UK).

Example 291-[2-O-(2,4,-dimethoxybenzoyl)-4-thio-β-D-ribofuranosyl]uracil (41)

Compound 12 (1 eq.) is dissolved in dry THF. TEA (5 eq.) and TREAT.HF(10 eq.) are added and the mixture is stirred at room temperature for 6h. The solvents are removed under reduced pressure and the residue isco-evaporated with toluene in vacuo. The residue is purified by flashchromatography to give Compound 41.

Example 301-[3,5-O-p-methoxybenzylidene-2-O-(2,4,-dimethoxybenzoyl)-4-thio-β-D-ribofuranosyl]uracil(42)

Compound 41 (1 eq.) is dissolved in dry DMF. Camphor sulfonic acid (0.2eq.), and p-methoxybenzaldehyde-dimethylacetal (4 eq.) are added and thereaction is stirred at room temperature until all starting material isconsumed. The reaction mixture is diluted with ethyl acetate and washedwith saturated sodium bicarbonate solution (2×), water (1×), brine (1×)and dried over anhydrous sodium sulfate. Following concentration underreduced pressure the residue is purified by flash chromatography to giveCompound 42.

Example 31 1-[3,5-Op-methoxybenzylidene-4-thio-β-D-ribofuranosyl]uracil(43)

Compound 42 is dissolved in methanolic ammonia and stirred at roomtemperature for 13 h. The reaction mixture is concentrated under reducedpressure and the residue is purified by flash chromatography to giveCompound 43.

Example 32 3′,5′-O-p-methoxybenzylidene-4′-thio-O²,2′-anhydrouridine(44)

Compound 43 (1 eq.) is treated with diphenylcarbonate (21.2 eq.) in dryDMF, heated to 90° C. and sodium bicarbonate is added (10 grams per gramof Compound 43). The reaction is held at 110° C. for 2.5 h., cooled andfiltered. The residue is washed several times with ethyl acetate and thewashings are combined with the initial filtrate. The combined filtratesare washed with water (2×), brine (1×), and dried over anhydrous sodiumsulfate. The solvents are removed under reduced pressure and the residueis purified by flash chromatography to give Compound 44.

Example 331-[3,5-O-p-methoxybenzylidene-2-fluoro-4-thio-β-D-ribofuranosyl]uracil(45)

Compound 44 (1 eq.) is dissolved in dry THF. TBAF (10 eq.) is added andthe reaction mixture is refluxed until starting material is consumed.The solvent is removed under reduced pressure and the residue purifiedby flash chromatography to give Compound 45.

Alternately Compound 45 is also obtained by heating Compound 44 in dryDMF in presence of 18-crown-6 (5 eq.) and KF (20 eq) at 110° C. followedby above work-up and flash chromatography.

Example 34 1-[2-fluoro-4-thio-β-D-ribofuranosyl]uracil (46)

Compound 45 is dissolved in 80% AcOH and stirred at room temperature for10 h. The reaction mixture is concentrated under reduced pressure andco-evaporated twice with toluene. The residue is purified by flashchromatography to give Compound 46.

Example 351-[3,5-O-p-methoxybenzylidene-4-thio-β-D-arabinofuranosyl]uracil (47)

Compound 44 (0.3 mmol) is dissolved in dioxane (2 mL) and a 2N NaOH soln(2 mL) is added. The reaction mixture is stirred at room temperaturetill all starting material is consumed. The reaction mixture isneutralized with acetic acid and concentrated to dryness under reducedpressure. The residue is purified by flash chromatography to giveCompound 47.

Example 361-[3,5-O-p-methoxybenzylidene-2-methanesulfonyl-4-thio-β-D-arabinofuranosyl]uracil(48)

Compound 47 (1 eq.) is dissolved in dry pyridine and cooled to 0° C.Methanesulfonyl chloride (1.5 eq.) is added and the reaction is stirredat room temperature over night. The reaction is quenched by pouring ontoice and the aqueous phase is extracted three times with dichloromethane.The dichloromethane solution is concentrated under reduced pressure andthe residue is purified by flash chromatography to give Compound 48.

Example 371-[3,5-O-p-methoxybenzylidene-2-fluoro-4-thio-β-D-ribofuranosyl]uracil(45)

Compound 48 (1 eq.) is dissolved in dry DMF. TBAF (10 eq.) is added andthe reaction mixture is heated at 100° C. until starting material isconsumed. The solvent is removed under reduced pressure and the residuepurified by flash chromatography to give Compound 45.

Alternately, Compound 45 is obtained by heating Compound 48 in dry DMFin presence of 18-crown-6 (5 eq.) and KF (20 eq) at 110° C. followed byabove work-up and flash chromatography.

Example 38 1-[2-fluoro-4-thio-β-D-ribofuranosyl]uracil (46)

Compound 45 is dissolved in 80% AcOH and stirred at room temperature for10 h. The reaction mixture is concentrated under reduced pressure andco-evaporated twice with toluene. The residue is purified by flashchromatography to give Compound 46.

Example 39 5′-O-DMT-2′-fluoro-2′-deoxy-4′-thio-uridine

The title compound is prepared using the procedures illustrated abovefor Compound 14.

Example 401-(3-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-5-O-(4,4′-dimethoxytrityl)-2-fluoro-2-deoxy)-4-thio-β-D-ribofuranosyl)uracil(49)

The title compound is prepared from5′-O-DMT-2′-fluoro-2′-deoxy-4′-thio-uridine using the proceduresillustrated above for Compound 16.

Example 415′-O-4,4′-Dimethoxytrityl)-2′-O-[2-(methoxy)ethyl]4′-thiouridine-3′-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite(53)

Compound 13 is heated with diphenylcarbonate and sodium bicarbonate at100° C. in dimethyl acetamide to yield Compound 50. Compound 50 onrefluxing with anhydrous 2-methoxyethanol and aluminium2-methoxyrthoxide will yield Compound 51. Compound 51 on selectivetritylation at 5′-position with DMTCI and in pyridine in presence ofcatalytic amount of DMAP will yield Compound 52, which is phosphitylatedat 3′-position to yield Compound 53.

Example 42N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-[2-(methoxy)ethyl]4′-thiocytidine-3′-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite(56)

The 3′-hydroxyl group of Compound 52 is transiently protected withtrimethylsilyl group on treatment with TMSCl in pyridine. Thistransiently protected compound is further treated with 1,2,4-triazoleand POCl₃ in acetonitrile to give the triazole derivative at 4-positionof the base. The triazolide is treated with ammonium hydroxide indioxane to give Compound 54 which is converted to Compound 55 bytreatment with benzoic anhydride in DMF at room temperature.Phosphitylation of Compound 55 will yield Compound 56.

Example 435′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methoxy)ethyl]-N²-isobutyryl-4′-thioguanosine-3′-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite(66)

Synthesis of compound 66 is described in Scheme 13. Compound 61 ontreatment with Cs₂CO₃, and toluene-4-sulfonic acid 2-methoxy-ethyl esterin DMF will give Compound 62. Treatment of Compound 62 with adenosinedeaminase in phosphate buffer at pH 7.5 will yield Compound 63. Theexocyclic amino group of Compound 63 is protected by treatment withisobutyryl group under transient protection conditions to yield Compound64. Compound 64 is treated with DMTCI in pyridine at room temperature inthe presence of catalytic amount of DMAP to yield Compound 65.Phosphitylation of Compound 65 will give Compound 66.

Example 445′-O-(4,4′-dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-O-succinyl-4′-thiouridine(67)

Compound 15b (0.26 g, 0.38 mmol) was mixed with succinic anhydride (0.06g, 0.57 mmol) and DMAP (0.02 g, 0.2 mmol) and dried under reducedpressure at 40° C. overnight. The mixture was dissolved in anhydrousClCH₂—CH₂Cl (1 mL) and triethyl amine (0.11 mL, 0.8 mmol) was added. Thesolution stirred at room temperature under argon atmosphere for 7 h. Itwas then diluted with CH₂Cl₂ (20 mL) and washed with ice cold aqueouscitric acid (10 wt %, 20 mL) and brine (20 mL). The organic phase wasdried over anhydrous Na₂SO₄ and concentrated to dryness. The residuethus obtained was purified by flash column chromatography on silica-gel.The column was eluted with 10% MeOH in CH₂Cl₂ to afford the titlecompound in 83% isolated yield (0.24 g): R_(f) 0.1 (50% ethyl acetate inhexane).

¹H NMR (200 MHz, CDCl₃) δ 0.06 (s, 3H), 0.07 (s, 3H), 0.91 (s, 9H),2.45-2.67 (m, 4H), 3.35-3.50 (m, 2H), 3.79 (s, 6H), 4.32 (d, J=3.2 Hz,1H), 5.19 (dd, J=3.2 and 5.6 Hz, 1H), 5.55 (d, J=8.20 Hz, 1H), 6.45 (d,J=8.80 Hz, 4H), 6.84 (d, J=8.8 Hz, 1H), 7.23-7.45 (m, 9H), 7.59 (d,J=8.2 Hz, 1H), 10.12 (br s, 1H); ¹³C NMR (50 MHz, CDCl₃) δ −4.3, −4.8,25.5, 28.5, 29.0, 55.1, 60.6, 60.3, 64.9, 73.6, 77.4, 87.2, 103.2,113.2, 127.0, 127.9, 128.1, 130.1, 135.0, 135.3, 140.8, 144.2, 151.3,158.6, 163.8, 171.3, 175.7; MS (FAB) m/z 799.20 [M+Na]⁺.

Example 455′-O-(4,4′-dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-4′-thiouridine-2′-O-succinylCPG (68)

Compound 67 was loaded on to the aminoalkyl controlled pore glass (CPG)according to the standard synthetic procedure (TBTU mediated synthesisof functionalized CPG synthesis: Bayer, E.; Bleicher, K.; Maier, M. A.;Z. Naturforsch. 1995, 50b, 1096-1100) to yield the functionalized solidsupport 68 (49 μmol/g).

Example 465′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methoxy)ethyl]-N⁶-benzoyl-4′-thioadenosine-3′-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite(69)

Compound 69 is prepared by first treating 4′-thioadenosine (Example 24)with Cs₂CO₃, and toluene-4-sulfonic acid 2-methoxy-ethyl ester in DMF toprovide the 2′-O-methoxyethyl intermediate. This material is furthertransiently protected with TMSCl in pyridine and is further treated withbenzoyl chloride to give the N6-Bz protected intermediate. The5′-hydroxyl group of the N6-Bz protected intermediate is selectivelyprotected using dimethoxytritylchloride. The DMT protected compound isphosphitylated under standard conditions using2-cyanoethoxy-N,N,N′,N′-tetraisopropylphosphoramidite as previouslydescribed as per example 16 above to give the title compound.

Example 47 4′-Thio Modified RNA Synthesis

Oligonucleotides with 4′-thio modifications were synthesized. 0.1 Msolution of amidites in anhydrous acetonitrile was used for thesynthesis of the modified oligonucleotides. The phosphoramiditesolutions were delivered in two portions, each followed by a 5 min.coupling wait time. The standard 2′-O-TBDMS phosphoramidites andcommercial solid supports (Glen Research Inc.) were used for theincorporation of A, C, G and U residues. Oxidation of theinternucleotide phosphite triester to phosphate triester was carried outusing tert-butylhydroperoxide/acetonitrile/water (10:87:3) with a waittime of 10 min. All other steps in the protocol supplied by Milliporewere used without modifications. The coupling efficiencies were morethan 97%. After completion of the synthesis, CPG was suspended inaqueous ammonium hydroxide (30 wt. %):ethanol (2:1) and kept at roomtemperature for 2 h. The CPG was filtered and the filtreate was heatedat 55° C. for 6 h to complete the removal of all protecting groupsexcept the TBDMS group at 2′-position. The residue obtained wasre-suspended in anhydrous TEA.HF/NMP solution (1 mL of a solution of 1.5mL N-methylpyrrolidine, 750 μl TEA and 1 ml of TEA3HF to provide a 1.4 MHF concentration) and heated at 65° C. for 1.5 h to remove the TBDMSgroups at 2′-position. The reaction was quenched with 1.5 M ammoniumbicarbonate (1 mL) and the mixture was loaded on to a Sephadex G-25column (NAP Columns, Amersham Biosciences Inc.). The oligonucleotideswere eluted with water and the fractions containing the oligonucleotideswere pooled together and purified by High Performance LiquidChromatography (HPLC, Waters, C-18, 7.8×300 mm, A=100 mMtriethylammonium acetate, pH=7, B=acetonitrile, 5 to 20% B in 40 min,then 60% Bin 60 min, Flow 2.5 mL min⁻¹, λ=260 nm). Fractions containingfull-length oligonucleotides were pooled together (assessed by CGEanalysis >90%) and evaporated. The residue was dissolved in sterile 10 Mammonium acetate (0.3 mL) solution. Ethanol (1 mL) was added and cooledto −78° C. for 1 h to get a precipitate and pelleted out the precipitatein a microfuge (NYCentrifuge 5415C; Eppendorf, Westbury, N.Y.) at 3000rpm (735 g) for 15 min. The pellets were collected by decanting thesupernatant. The pelleted oligonucleotides are dissolved in sterilewater (0.3 mL) and precipitated by addition of ethanol (1 mL) andcooling the mixture at −78° C. for 1 h. The precipitate formed waspelleted out and collected as described above. The isolated yields formodified oligonucleotides were 30-35%. The oligonucleotides werecharacterized by ES MS analysis and purity was assessed by capillary gelelectrophoresis and HPLC (Waters, C-18, 3.9×300 mm, A=100 mMtriethylammonium acetate, pH=7, B=acetonitrile, 5 to 60% B in 40 min,Flow 1.5 mL min⁻¹, λ=260 nm).

Example 48 Synthesis of 2′-OMe-4′-thio-U phosphoramidite (40)

2,2′-Anhydro-4′-Thio-Uridine (50)

4′-Thio-uridine (13) (1 gm, 3.8 mmole), sodium bicarbonate (5 mg),diphenyl carbonate (900 mg, 4.0 mmol) were dissolved in 10 mL dryN,N-dimethylacetamide and heated at 100° C. for 2 hrs. The reactionmixture was cooled to room temperature and poured into rapidly stirringether. The resulting precipitate was isolated by centrifugation (800 mg,76% yield) and used as such in the next step. 1H NMR (DMSO-d6): δ 7.8(d, 1H), 6.2 (d, 1H), 5.82 (m, 2H), 5.4 (d, 1H), 5.2 (m, 1H), 4.6 (m,1H), 3.2-3.4 (m, 3H). ESMS=243 (MH⁺), calc for C₉H₁₀N₂O₄S=242.03.

2′-O-methyl-4′-thio-Uridine (38)

Compound 50 (1 gm, 4.0 mmole), trimethyl borate (2 mL), trimethylorthoformate (1 mL), sodium bicarbonate (5 mg) and methanol (3 mL) wereadded to a steel bomb and heated at 150° C. for 2 days. The bomb wascooled to room temperature and the residue was concentrated underreduced pressure and coevaporated several times with methanol. Theresidue was purified by flash chromatography (2% methanol indichloromethane) to give 38 (800 mg, 75% yield). 1H NMR (DMSO-d6): δ 8.0(d, 1H), 5.95 (d, 1H), 5.6 (d, 1H), 5.15-5.25 (br, 2H), 4.2 (m, 1H),3.82 (m, 1H), 3.62 (m, 2H), 3.5 (s, 3H), 3.38 (m, 1H). ESMS=275 (MH⁺),calc for C10H14N2O5S=274.06.

5′-(4,4′-dimethoxytrityl)-2′-O-methyl-4′-thio-uridine (39)

Compound 38 (400 mg, 1.46 mmole) was dissolved in cold, dry pyridine.Dimethoxytrityl chloride (600 mg, 1.752 mmole) was added and the mixturewas stirred at room temperature for 24 hrs. Solvents were removed underreduced pressure and the residue was purified by flash chromatography(1:1 ethyl acetate: dichloromethane) to give 39 (600 mg, 75% yield). ¹HNMR CDCl₃: δ 8.05 (d, 1H), 7.15-7.45 (m, 9H), 6.85 (m, 4H), 6.05 (d,1H), 5.45 (d, 1H), 4.2 (br, 1H), 3.8 (s, 7H), 3.62 (m, 2H), 3.55 (s,3H), 3.4 (br, 1H). FAB MS=577.2 (MH⁺), calc for C31H32N2O7S=576.19.

3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]-5′-(4,4′-dimethoxytrityl)-2′-O-methyl-4′-thio-uridine(40)

Compound 39 (400 mg, 0.7 mmole) was treated with2-cyanoethyl-N,N-diisopropyl chloro phosphoramidite (125 μL) anddiisopropylethylamine (200 μL) in dry dichloromethane (5 mL) for 2 hrsat 0° C. The reaction mixture was separated between ethyl acetate (50mL) and saturated sodium bicarbonate solution (20 mL). The ethyl acetatelayer was washed twice with saturated sodium bicarbonate solution (20mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by flash chromatography (10% acetonein methylene chloride) to give 40 (400 mg, 60% yield). 1H NMR (CDCl3): δ8.1 (d, 0.5H), 7.9 (d, 0.5H), 7.2-7.5 (m, 10H), 6.85 (m, 4H), 6.05 (dd,1H), 5.55 (dd, 1H), 4.3 (m, 1H), 3.8 (m, 7H), 3.55 (m, 5H), 2.6 (m,1.2H), 2.4 (m, 0.8H), 1.0-1.2 (m, 12H). FABMS=777.300 (MH⁺), calc forC40H49N4O8PS=776.3009.

Example 49 Synthesis of 2′-OMe-4′-Thio-C Phosphoramidite

1-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-O-methyl-4-thio-β-D-ribofuranosyl]uracil(108)

Compound 38 (70 mg, 0.26 mmole) was dissolved in anhydrous pyridine (3mL) under a nitrogen atmosphere and cooled in an ice bath. TIPDS-Cl₂(166 μL, 0.52 mmole) was added drop-wise with vigorous stirring.Stirring was continued at the same temperature for an additional 2 to 4h. When all of 38 had been consumed the reaction was quenched by pouringonto ice. The mixture was separated between ethyl acetate and water andthe ethyl acetate layer was washed thrice with cold sat. sodiumbicarbonate solution and once with brine. The ethyl acetate layer wasdried over anhydrous sodium sulfate and concentrated under reducedpressure. The crude material was purified by flash chromatography (5%methanol in dichloromethane) to give Compound 108 (120 mg, 90% yield).1H NMR (CDCl3): δ 8.02 (d, 1H), 6.02 (d, 1H), 5.6 (d, 1H), 4.2 (br, 1H),3.62 (m, 3H), 3.55 (s, 3H), 3.4 (br, 1H), 1.1-0.9 (m, 28H). ESMS=517(MH⁺), calc for C₂₂H₄₀N₂O₆SSi₂=516.02.

1-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-O-methyl-4-thio-β-D-ribofuranosyl]-N-benzoyl-cytosine(109)

Compound 108 (475 mg, 0.92 mmole) was dissolved in dry acetonitrile (4mL). In a separate flask 1,2,4-triazole (0.89 g, 12.88 mmole) wassuspended in dry acetonitrile (12 mL), under nitrogen and cooled to 0°C. in an ice-bath. POCl₃ (0.33 mL, 3.68 mmole) was added dropwise withvigorous stirring, followed by dropwise addition of TEA (2.6 mL, 18.4mmole) in acetonitrile (6 mL). After TEA addition was complete thereaction mixture was stirred at the same temperature for an additional30 min. The solution of 108 was added and the reaction stirred at 0° C.for 1 hr. and then at room temperature for 12 hrs. At the end of thisperiod the crude reaction was separated between ethyl acetate and sat.bicarbonate solution. The ethyl acetate layer was washed twice with sat.sodium bicarbonate solution and once with brine. After drying overanhydrous sodium sulfate the solvents were removed under reducedpressure. The resulting oily residue was taken up in 1,4-dioxane (10 mL)and aqueous ammonia solution (5 mL) was added. The reaction was stirredin a sealed flask for 12 hrs. The reaction mixture was separated betweenethyl acetate and water. The ethyl acetate layer was washed twice withwater and once with brine, dried over anhydrous sodium sulfate, andconcentration under reduced pressure. The resulting crude residue wasdissolved in dry acetonitrile (220 mg, 0.43 mmole).Dimethylaminopyridine (DMAP) (55 mg, 0.43 mmole) and benzoic anhydride(150 mg, 0.853 mmole) were added and the reaction was stirred at 65° C.for 20 min. The reaction was partitioned between ethyl acetate and waterand the ethyl acetate layer was washed thrice with sat. sodiumbicarbonate solution, once with brine, and dried over anhydrous sodiumsulfate. Solvents were removed under reduced pressure and the residuewas purified by flash chromatography (50% ethyl acetate indichloromethane) to give Compound 109 (480 mg, 85% yield). 1H NMR(CDCl3): δ 8.9 (d, 1H), 8.7 (br s, 1H), 7.9 (d, 2H), 7.5 (m, 3H), 5.96(s, 1H), 4.15 (m, 3H), 3.85 (m, 5H), 1.15-0.8 (m, 28H). ESMS=620 (MH⁺)calc for C₂₉H₄₅N₃O₆SSi₂=619.2.

1-[5-(4,4′-dimethoxytrityl)-2-O-methyl-4-thio-β-D-ribofuranosyl]-N-benzoyl-cytosine(110)

Compound 109 (200 mg, 0.32 mmole) was dissolved in dry THF. TREAT.HF(1.5 mL) and TEA (0.75 mL) were added and the mixture stirred for 4 hrs.The solvent was removed under reduced pressure followed by twoco-evaporations with dry toluene. The residue was further coevaporatedtwice with anhydrous pyridine (10 mL) and dried overnight overphosphorous pentoxide. The residue was dissolved in cold, dry pyridine(5 mL). DMAP (8 mg, 0.07 mmole) and dimethoxytrityl chloride (200 mg,0.6 mmole) were added and the mixture was stirred at room temperaturefor 24 hrs. The residue purified by flash chromatography (10% methanolin dichloromethane) to give Compound 110 (180 mg, 80% yield). 1H NMR(CDCl3): δ 8.8 (d, 1H), 8.6 (d, 1H), 7.9 (d, 2H), 7.6-7.2 (m, 13H), 6.9(d, 4H), 6.15 (s, 1H), 4.2 (br, 1H), 3.8 (s, 6H), 3.76 (m, 1H), 3.73 (s,3H), 3.66 (m, 3H). FABMS=680.2 (MH⁺), cal for C₃₈H₃₇N₃O₇S=679.235.

3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]-5′-(4,4′-dimethoxytrityl)-2′-O-methyl-4′-thio-N-benzoyl-cytidine(111)

Compound 110 (190 mg) was treated with 2-cyanoethyl-N,N-diisopropylchloro phosphoramidite (125 μL) and diisopropylethylamine (200 μL) indry dichloromethane (5 mL) for 2 hrs at 0° C. The reaction mixture wasseparated between ethyl acetate (50 mL) and saturated sodium bicarbonatesolution (20 mL). The ethyl acetate layer was washed twice withsaturated sodium bicarbonate solution (20 mL), dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by flash chromatography (30% ethyl acetate in hexanes) to give113 (150 mg, 75% yield). 1H NMR (CDCl₃): δ 8.8 (d, 0.5H), 8.6 (d, 0.5H),7.9 (d, 2H), 6.9 d(4H), 7.6-7.2 (15H), 6.1 (d, 1H), 4.4 (m, 0.5H), 4.2(m, 0.5H), 3.9 (m, 1H), 3.8 (d, 6H), 3.7-3.3 (m, 7H), 2.55 (m, 0.5H),2.4 (m, 0.5H), 1.3-1.0 (m, 12H). FABMS=880.3 (MH⁺), calc forC₄₇H₅₄N₅O₈PS=879.3431.

Example 50 Synthesis of 2′-O-methyl-4′-thio-modified siRNA

The standard phosphoramidites and solid supports were used forincorporation of A, U, G, and C residues. A 0.1 M solution of theamidites in anhydrous acetonitrile was used for the synthesis. Chemicalphosphorylation reagent procured form Glen Research Inc., Virginia, USAwas used to phosphorylate the 5′-terminus of modified oligonucleotides.The modified oligonucleotides were synthesized on functionalizedcontrolled pore glass (CPG) on an automated solid phase DNA synthesizer.The universal solid support was used for the synthesis ofoligonucleotides bearing a terminal 2′-deoxy-2′-fluoro modification.⁴³Twelve equivalents of phosphoramidite solutions were delivered in twoportions, each followed by a 6 min coupling wait time. All other stepsin the protocol supplied by the manufacturer were used withoutmodification. A solution of tert-butyl hydroperoxide/acetonitrile/water(10:87:3) was used to oxidize inter nucleosidic phosphite to phosphate.The coupling efficiencies were more than 97%. After completion of thesynthesis, solid support was suspended in aqueous ammonium hydroxide (30wt. %): ethanol (3:1) and heated at 55° C. for 6 h to complete theremoval of all protecting groups except TBDMS group at 2′-position. Thesolid support was filtered and the filtrate was concentrated to dryness.The residue obtained was re-suspended in anhydrous triethylaminetrihydrofluoride/triethylamine/1-methyl-2-pyrrolidinone solution (0.75mL of a solution of 1 ml of triethyl amine trihydofluoride, 750 μltriethylamine and 1.5 mL 1-methyl-2-pyrrolidine, to provide a 1.4 M HFconcentration) and heated at 65° C. for 1.5 h to remove the TBDMS groupsat the 2′-position.³⁷ The reaction was quenched with 1.5 M ammoniumbicarbonate (0.75 mL) and the mixture was loaded on to a Sephadex G-25column (NAP Columns, Amersham Biosciences Inc.). The oligonucleotideswere eluted with water and the fractions containing the oligonucleotideswere pooled together and purified by High Performance LiquidChromatography (HPLC) on a strong anion exchange column (Mono Q,Pharmacia Biotech, 16/10, 20 mL, 10 μm, ionic capacity 0.27-0.37mmol/mL, A=100 mM ammonium acetate, 30% aqueous acetonitrile, B=1.5 MNaBr in A, 0 to 60% B in 40 min, Flow 1.5 mL min⁻¹, λ=260 nm). Fractionscontaining full-length oligonucleotides were pooled together (assessedby CGE analysis >90%) and evaporated. The residue was dissolved insterile water (0.3 mL) and absolute ethanol (1 mL) was added and cooledin dry ice (−78° C.) for 1 h and the precipitate formed was pelleted outby centrifugation (NYCentrifuge 5415C; Eppendorf, Westbury, N.Y.) at3000 rpm (735 g). The supernatant was decanted and the pellet wasre-dissolved in 10 M ammonium acetate (0.3 mL) solution. Ethanol (1 mL)was added and cooled to −78° C. for 1 h to get a precipitate andpelleted out the precipitate in a centrifuge (NYCentrifuge 5415C;Eppendorf, Westbury, N.Y.) at 3000-rpm (735 g) for 15 min. The pelletwas collected by decanting the supernatant. Re-dissolved the pelletedoligonucleotides in sterile water (0.3 mL) and precipitated by addingethanol (1 mL) and cooling the mixture at −78° C. for 1 h. Theprecipitate formed was pelleted out and collected as described above.The oligonucleotides were characterized by ES MS and purity was assessedby capillary gel electrophoresis and HPLC (Waters, C-18, 3.9×300 mm,A=100 mM triethylammonium acetate, pH=7, B=acetonitrile, 5 to 60% B in40 min, Flow 1.5 mL min⁻¹, λ=260 nm).

TABLE 2 2′-O-methyl-4′-thio modified antisense strand of siRNA targetedto PTEN mRNA ISIS Found No. Construct Cald Mass Mass 3757623′-UU*CAUUC*CUGGUCUCUG⁹ 5995.01 5994.40 UU-5′ 3757613′-U*U*C*AUUCCUGGUCUCUG 6013.01 6012.01 U^(σ)U^(σ)-5′ U*= 2′-O-methyl-4′-thiouridine, C* = 2′-O-methyl-4′-thiocytidine, G⁹= 2′-O-methyl guanosine, U^(σ )= 4′-thiouridine

Example 51 Synthesis of 2′-O-(2-Methoxyethyl)-4′-thio-uridine

5′-O-tert-butyldiphenylsilyl-4-thiouridine (112)

4′-thio-uridine (2.95 g, 11.43 mmol) was mixed with DMAP (0.02 g, 0.15mmol) and dried under reduced pressure over P₂O₅. The reaction mixturewas dissolved in anhydrous pyridine (15 mL) and tert.-butyldiphenylsilylchloride (3.27 mL, 13 mmol) was added. Stirred the reaction mixture atroom temperature under argon atmosphere for 12 h. The solvent wasremoved under reduced pressure residue dissolved in ethyl acetate (100mL). The organic phase washed with aqueous NaHCO₃ (5%, 50 mL), brine (50mL). Organic layer separated and dried over anhydrous Na2SO4 andconcentrated under reduced pressure. The residue obtained was purifiedby flash silica gel column chromatography and eluted withdichloromethane containing in cremental amount odf methanol (5-10%) toyield Compound 112 (5.21 g, 91%) as a foam. ¹H NMR (200 MHz, DMSO-d₆):11.33 (s, 1H), 7.84 (d, J=8.2 Hz, 1H,), 7.66-7.33 (m, 10H), 5.86 (d,J=6.4 Hz, 1H), 5.58 (d, J=5.6 Hz, 1H), 5.47 (d, J=8.2 Hz, 1H), 5.34 (d,J=4.4 Hz, 1H), 4.09 (m, 2H), 3.97 (m, 1H), 3.77 (m, 1H), 3.35 (m, 1H),1.01 (s, 9H); MS (API-ES) m/z 499.1 [M+H]⁺.

2,2′-Anhydro-5′-O-tert-butyldiphenylsilyl-4′-thio-uridine (113)

To a dried mixture of Compound 112 (1.1 g, 2.20 mmol), diphenylcarbonate(0.55 g, 2.42 mmol) and anhydrous NaHCO₃ (73.7 mg, 0.88 mmol) dimethylacetamide (5.5 mL) was added. The reaction mixture was heated at 100° C.5 h. The solvent was distilled out under reduced pressure to get an oilsand the oil was purified by falsh silica gel column chromatography andeluted with 5 to 10% MeOH in dichloromethane to yield Compound 113 (0.87g, 82%). ¹H NMR (200 MHz, DMSO-d₆): 7.81 (d, J=7.4 Hz, 1H,), 7.57-7.34(m, 10H), 6.19 (d, J=7.4 Hz, 1H), 5.98 (d, J=4.4 Hz, 1H), 5.39 (d, 1H),5.58 (d, J=5.6 Hz, 1H), 4.76 (br s, 1H), 3.40-3.7 (m, 3H), 1.00 (s, 9H);MS (API-ES) m/z 481.1 [M+H]⁺.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-methoxyethyl)-4′-thio-uridine (114)

Compound 113 (0.7 g, 1.45 mmol) was mixed withtris(2-methoxyethyl)borate (0.63 g, 2.93 mmol), NaHCO₃ (0.02 g, 0.23mmol) and 2-methoxyethanol (7 mL). The mixture was heated at 140° C. for21 h. The solvent was removed under reduced pressure and to the residuewater (5 mL) and concentrated the solvent on a rotavapour keeping waterbath temperature 50-60° C. Repeated this process twice. Residue obtainedwas purified by falsh silica gel column chromatography and eluted with5% MeOH in CH₂Cl₂ to yield Compound 114 (0.51 g, 66%0 as a foam. ¹H NMR(200 MHz, DMSO-d₆): 11.38 (s, 1H), 7.89 (d, 1H,), 7.67-7.44 (m, 10H),5.92 (d, 1H), 5.69 (d, 1H), 5.46 (d, 1H), 5.30 (d, 1H), 4.17 (m, 1H),4.01-3.94 (m, 2H), 3.82 (m, 1H), 3.58 (m, 1H), 3.52-3.38 (m, 4H), 3.20(s, 3H), 1.01 (s, 9H); MS (API-ES) m/z 579.1 [M+Na]⁺.

2′-O-(2-Methoxyethyl)-4′-thio-uridine (51)

To a solution of Compound 114 (0.49 g, 0.88 mmol) in anhydrous THF (37mL) 1M tetrabutylammonium fluoride in THF (1.8 mL) was added. Aceticacid (0.12 mL, 2.03 mmol) was added to this mixture and the resultingreaction mixture stirred at room temperature for 30 min. The solvent wasremoved under reduced pressure and the residue obtained was purified byflash silica gel column chromatography and eluted with acetone to yieldCompound 51 (0.23 g, 83%). ¹H NMR (300 MHz, DMSO-d₆): 11.2 (s, 1H), 8.04(d, 1H,), 5.94 (d, 1H), 5.69 (d, 1H), 5.20 (d, 1H), 4.15 (t, 1H), 4.04(m, 1H), 3.67-3.53 (m, 6H), 3.47-3.36 (m, 2H), 3.31 (s, 3H); MS (API-ES)m/z 341.0 [M+Na]⁺.

2′-O-(2-Methoxyethyl)-4′-thio-uridine (52)

Compound 51 (0.27, 0.85 mmol) was mixed with DMAP (0.05 g, 0.43 mmol)and dried under reduced pressure. The mixture was dissolved in anhydrouspyridine and 4,4′-dimethoxytrityl chloride (0.36 g, 1.06 mmol) was addedand the resulting solution stirred at room temperature under inertatmosphere for 20 h. Solvent was removed under reduced pressure and theresidue obtained was dissolved in dichloromethane (50 ml) and washedwith aqueous NaHCO₃ (5 wt %, 40 mL) and brine (40 mL). The organic phasedried over anhydrous Na₂SO₄ and evaporated under reduced pressure. Theresidue obtained was purified by flash silica gel column chromatographyand eluted with 0-5% MeOH in CH₂Cl₂ to yield Compound 52 (0.32 g, 61%).¹H NMR (200 MHz, DMSO-d₆): 11.39 (s, 1H), 7.76 (d, 1H), 7.74-7.15 (m,9H) 6.90 (d, 4H), 5.89 (d, 1H), 5.50 (dd, 1H), 5.25 (d, 1H), 4.05 (m,1H), 3.88 (m, 1H), 3.73 (s, 6H), 3.53 (m, 1H), 3.48-3.53 (m, 5H), 3.18(s, 3H); MS (API-ES) m/z 643.2 [M+Na]⁺.

Example 52 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy(2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 53 Oligonucleotide and Oligonucleoside Synthesis

The antisense oligomeric compounds used in accordance with thisinvention may be conveniently and routinely made through the well-knowntechnique of solid phase synthesis. Equipment for such synthesis is soldby several vendors including, for example, Applied Biosystems (FosterCity, Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages are prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289,all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 54 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense oligomeric compounds (RNA oligonucleotides) of the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA antisense oligomeric compounds can then be annealed bymethods known in the art to form double stranded (duplexed) antisenseoligomeric compounds. For example, duplexes can be formed by combining30 μl of each of the complementary strands of RNA oligonucleotides (50uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate)followed by heating for 1 minute at 90° C., then 1 hour at 37° C. Theresulting duplexed antisense oligomeric compounds can be used in kits,assays, screens, or other methods to investigate the role of a targetnucleic acid.

Example 55 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 56 Design and Screening of Duplexed Antisense OligomericCompounds Directed to a Selected Target

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense oligomeric compounds of the presentinvention and their complements can be designed to target a target. Theends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense oligomeric compounds are evaluatedfor their ability to modulate a target expression.

When cells reached 80% confluency, they are treated with duplexedantisense oligomeric compounds of the invention. For cells grown in96-well plates, wells are washed once with 200 μL OPTI-MEM-1reduced-serum medium (Gibco BRL) and then treated with 130 μL ofOPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desiredduplex antisense oligomeric compound at a final concentration of 200 nM.After 5 hours of treatment, the medium is replaced with fresh medium.Cells are harvested 16 hours after treatment, at which time RNA isisolated and target reduction measured by RT-PCR.

Example 57 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32 +/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 58 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 59 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates were diluted fromthe master plate using single and multi-channel robotic pipettors.Plates were judged to be acceptable if at least 85% of the oligomericcompounds on the plate were at least 85% full length.

Example 60 Cell Culture and Oligonucleotide Treatment

The effect of antisense oligomeric compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide. A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier. HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Oligomeric Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO:5) which is targeted to human H-ras, orISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:6) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO:7, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 61 Analysis of Oligonucleotide Inhibition of a Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently desired. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. One method of RNA analysis of the presentinvention is the use of total cellular RNA as described in otherexamples herein. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 62 Design of Phenotypic Assays and In Vivo Studies for the Useof a Target Inhibitors

Phenotypic Assays

Once a target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the a target inhibitors.Hallmark genes, or those genes suspected to be associated with aspecific disease state, condition, or phenotype, are measured in bothtreated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study.

To account for the psychological effects of receiving treatments,volunteers are randomly given placebo or a target inhibitor.Furthermore, to prevent the doctors from being biased in treatments,they are not informed as to whether the medication they areadministering is a target inhibitor or a placebo. Using thisrandomization approach, each volunteer has the same chance of beinggiven either the new treatment or the placebo.

Volunteers receive either the a target inhibitor or placebo for eightweek period with biological parameters associated with the indicateddisease state or condition being measured at the beginning (baselinemeasurements before any treatment), end (after the final treatment), andat regular intervals during the study period. Such measurements includethe levels of nucleic acid molecules encoding a target or a targetprotein levels in body fluids, tissues or organs compared topre-treatment levels. Other measurements include, but are not limitedto, indices of the disease state or condition being treated, bodyweight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements. Information recorded for eachpatient includes age (years), gender, height (cm), family history ofdisease state or condition (yes/no), motivation rating(some/moderate/great) and number and type of previous treatment regimensfor the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and a target inhibitor treatment. In general,the volunteers treated with placebo have little or no response totreatment, whereas the volunteers treated with the a target inhibitorshow positive trends in their disease state or condition index at theconclusion of the study.

Example 63 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 64 Real-Time Quantitative PCR Analysis of a Target mRNA Levels

Quantitation of a target mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 mL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and are designed to hybridize to a human a target sequence, usingpublished sequence information.

Example 65 Northern Blot Analysis of a Target mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human a target, a human a target specific primer probe set isprepared by PCR To normalize for variations in loading and transferefficiency membranes are stripped and probed for humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, PaloAlto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 66 Antisense Inhibition of Human a Target Expression byOligonucleotides

In accordance with the present invention, a series of oligomericcompounds are designed to target different regions of the human targetRNA. The oligomeric compounds are analyzed for their effect on humantarget mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments. The targetregions to which these sequences are complementary are herein referredto as “suitable target segments” and are therefore suitable fortargeting by oligomeric compounds of the present invention. Thesequences represent the reverse complement of the suitable antisenseoligomeric compounds.

As these “suitable target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisenseoligomeric compounds of the present invention, one of skill in the artwill recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother oligomeric compounds that specifically hybridize to these suitabletarget segments and consequently inhibit the expression of a target.

According to the present invention, antisense oligomeric compoundsinclude antisense oligomeric compounds, antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, alternatesplicers, primers, probes, and other short oligomeric compounds whichhybridize to at least a portion of the target nucleic acid.

Example 67 Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Although the invention has been described in detail with respect tovarious preferred embodiments it is not intended to be limited thereto,but rather those skilled in the art will recognize that variations andmodifications may be made therein which are within the spirit of theinvention and the scope of the appended claims.

All publications, patents, published patent applications, and otherreferences mentioned herein are hereby incorporated by reference intheir entirety.

1. A compound of formula (I):

wherein: T₁ is H or a protecting group; T₂ is a phosphoramidite group;Bx is hydrogen or a nucleobase; X is fluoro, substituted orunsubstituted —O—C₁-C₁₂ alkyl, substituted or unsubstituted —O—C₂-C₁₂alkenyl, or substituted or unsubstituted —O—C₂-C₁₂ alkynyl.
 2. Thecompound of claim 1, wherein X is a group of formula Ia:

wherein: R_(b) is O; R_(d) is a single bond, O, S or C(═O); R_(e) isC₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)), N═C(R_(p))(R_(q)),N═C(R_(p))(R_(r)) or has formula Ic;

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl; R_(r)is —R_(x)—R_(y); each R_(s), R_(t), R_(u) and R_(v) is, independently,hydrogen, C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, substituted orunsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemicalfunctional group or a conjugate group, wherein the substituent groupsare selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; oroptionally, R_(u) and R_(v), together form a phthalimido moiety with thenitrogen atom to which they are attached; each R_(w) is, independently,substituted or unsubstituted C₁-C₁₀ alkyl, trifluoromethyl,cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy,2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl,iso-butyryl, phenyl or aryl; R_(k) is hydrogen, an amino protectinggroup or —R_(x)—R_(y); R_(x) is a bond or a linking moiety; R_(y) is achemical functional group, a conjugate group or a solid support medium;each R_(m) and R_(n) is, independently, H, an amino protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein thesubstituent groups are selected from hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyland alkynyl; or R_(m) and R_(n), together, are an amino protectinggroup, are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O or are a chemical functionalgroup; m_(a) is 1 to about 10; each mb is, independently, 0 or 1; mc is0 or an integer from 1 to 10; m_(d) is an integer from 1 to 10; andprovided that when mc is 0, and is greater than
 1. 3. The compound ofclaim 1, wherein X is selected from fluoro or substituted orunsubstituted —O—C₁-C₁₂ alkyl.
 4. The compound of claim 1, wherein X isselected from fluoro, —O—CH₃, or —O—CH₂CH₂—O—CH₃.
 5. The compound ofclaim 1, wherein X is O-allyl, O—(CH₂)_(ma)—O—N(R_(m))(R_(n)) orO—CH₂—C(═O)—N(R_(m))(R_(n)); wherein: each R_(m) and R_(n) is,independently, H, an amino protecting group, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl; and ma is from 1 to about10.
 6. The compound of claim 1, wherein said phosphoramidite group is—P[N[(CH(CH₃)₂]₂)O(CH₂)₂CN.